JOURNAL OF AVIAN BIOLOGY 37: 609
616, 2006

Comparing plumage colour measurements obtained directly from live birds and from collected feathers: the case of the great tit Parus major Javier Quesada and Juan Carlos Senar

Quesada, J. and Senar, J. C. 2006. Comparing plumage colour measurements obtained directly from live birds and from collected feathers: the case of the great tit Parus major.
J. Avian Biol. 37: 609
616. Birds frequently display a colourful plumage which is important both in inter and intraespecific communication, and either in sexual and social contexts. In last years some methodologies have been developed to analyse plumage coloration, but the use of the spectrometers has been particularly important for UV range. Measurement of plumage coloration with the spectrometer may be taken directly on the bird or, alternatively by collecting some feathers and measuring them later in the laboratory. However, few is known about the reliability of measures obtained from feathers and whether these are really representative of plumage coloration. We tested this assumption analysing measurements of carotenoids-based coloration components (lightness, chroma and hue) and lutein peak of the yellow breast of the great tit Parus major. We used two spectrometers (Ocean optics and Minolta) which calculate differently the colour components. Our results showed that direct measurement of bird was highly repeatable to determine lightness, chroma and hue for both spectrometers. Similar results we found for collected feathers procedure for both devices. Collected feathers provided high representative measurements of colour values with Minolta spectrometer. Lightness was highly repeatable when we used Ocean optic spectrometer, but chroma and hue were moderate. Lutein peak was also highly repeatable in all cases. The number of feathers used to measure plumage coloration in collected feathers procedure strongly influenced values of colour plumage variables. In general, values of lightness, chroma and hue stabilised when more than 10
15 feathers were used although we found slight differences between spectrometers. However, only four feathers were needed for lutein peak. Thus, our results stress the need to use a minimum number of feathers in measuring plumage coloration from collected feathers. J. Quesada (correspondence) and J. C. Senar, Unitat Associada d’Ecologia Evolutiva y de la Conducta (CSIC). Museu de Cie`ncies Naturals, Barcelona, Spain. Present address of J.Quesada: Laboratorio de Ecologı´a Funcional. Centro de Investigaciones en Ecosistemas. Ant. Ctra. Pa´tzcuaro No. 8701 Col. Ex-Hacienda de San Jose´ de La Huerta C.P. 58190 Morelia, Michoaca´n. Mexico. E-mail: [email protected]

Many species of birds frequently display a colourful plumage that conveys valuable information about the qualities of the bearer (Espmark et al. 2000, Hill 2002). Due to the importance of plumage coloration in inter and intraespecific communication and both in sexual and social contexts, it is of high relevance to have at disposal reliable and objective methods to measure plumage colour. In recent years different methodologies and approaches have been developed: Munsell tables (Peiponen 1992, Zuk and Decruyenaere 1994),

photographic analysis (Figuerola and Senar 2000, Fitze and Richner 2002, Kodric-Brown and Johnson 2002), digital colorimeters (Senar et al. 1998, Figuerola et al. 1999, Senar et al. 2003), or reflection spectrometers (Bennett et al. 1996, Hunt et al. 1997, Keyser and Hill 1999, Bright and Waas 2002, MacDougall and Montgomerie 2003, Doucet and Montgomerie 2003b, Alonso-Alvarez et al. 2004). Birds have a fourth pigment in the eye that allows them to see ultraviolet (UV) coloration and humans are

# JOURNAL OF AVIAN BIOLOGY JOURNAL OF AVIAN BIOLOGY 37:6 (2006)

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blind to this wavelength (Bennett et al. 1997, Andersson et al. 1998). Spectrometers have particularly turned out to be the only useful and precise enough tool to determine plumage coloration in this range of spectrum (300
400 nm; Andersson et al. 1998, Cuthill et al. 1999, Hunt et al. 1999, Siitari et al. 2002, Doucet and Montgomerie 2003a, Alonso-Alvarez et al. 2004), although it has also been used in the visible range (400
700 nm; Parker et al. 2003, Hill and Farmer 2005). Measurements of plumage coloration with the spectrometer may be taken directly on the bird (Andersson ¨ rnborg et al. 2002, et al. 1998, Siitari and Huhta 2002, O Eaton and Lanyon 2003, Doucet and Montgomerie 2003a, Alonso-Alvarez et al. 2004) but this requires to carry the devices to the field, which may be an important drawback because of the fragility of equipment (Berggren and Merila¨ 2004). A suitable alternative option is to collect some feathers and to measure them in the laboratory (Zuk and Decruyenaere 1994, Bennett et al. 1997, Keyser and Hill 1999, Perrier et al. 2002, Siefferman and Hill 2004). However, and in spite of the generalised use of spectrometers, very few is surprisingly known about the reliability of using one or the other alternative procedures. The aim of this study is: 1) to determine the reliability of measuring plumage coloration both directly on the animal in the field and from collected feathers in the laboratory, 2) to determine whether measurements from feathers in the laboratory are representative of the real plumage coloration measured directly on the animal, and 3) to determine if the number of feathers used in laboratory influences the final values of colour variables. Colour measurements were obtained from the great tit Parus major, a 18 g parid bird profusely used as a common biological model in plumage coloration studies (Cramp and Perrins 1994), which presents a ventral yellow coloration with a component of UV (Fig. 1), similarly to other species (MacDougall and Montgomerie 2003, Ha¨stad 2003).

Fig. 1. Spectrum of the great tit chest. The ventral yellow breast shows a peak of UV coloration similarly found to other species (see text for references).

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Materials and methods Field work was carried out during February 2004 in the field station of Sarria´ (Barcelona, North-eastern Spain). Sarria´ is a suburban area with orchards, almonds plantations and some scattered pieces of mixed haleppo pine Pinus halepensis and evergreen oak Quercus ilex forests. The use of a so short data collecting period (one month) allowed to avoid any possible colour degradation effect. We captured great tits in funnel baited traps (Senar et al. 1997), and then these were sexed and aged according to Svensson (1992). Colour measurements may be defined according to different systems (e.g. Munsell, CIE Yxy, CIE Lab, CIE LCH) but one of the most widely used is the CIE LCH colour system, which provides independent values of hue, chroma and lightness, the parameters generally used to define a colour. Hue corresponds to wavelength of light, chroma refers to spectral variance and therefore to colour purity so that the more monochromatic a colour is, the higher its chroma value. Lightness is correlated with physical light intensity and refers to the position on a grey-scale between black and white (Booth 1990, Dome`nech et al. 2000). Most devices calculate directly these three variables from the sample, but only considering the visible range (approx. 400
700 nm), so that this is the most used methodology to determine plumage colour in this part of the spectrum. Alternatively, since bird’s range of vision also encloses the UV spectrum, other methodologies have been used to measure lightness, chroma and hue from the values of reflectance spectrum given by spectrometers. Hence, in this work we considered the two methods of calculating the colour variables in order to get a wider generalisation of our results. Yellow plumage coloration was measured based on Eaton and Lanyon (2003) using a USB2000 spectrometer (Ocean Optics, Duiven, The Netherlands, EU) and a PX-2 pulsed xenon light (220
750 nm). All sample spectra were measured in reference to a white substance (WS-1, Diffuse Reflectance Standard) which has a reflectivity over 98% and a dark spectrum to eliminate noise given from spectrometer without any light reaching the light sensitive detector. We used some of the most common ways to calculate colour variables. The range of spectrum considered was limited between 300
700 nm (Hunt et al. 1999). For each individual spectrum we computed lightness as the mean of reflectance values in the region of the spectrum (Andersson et al. 1998, Doucet and Montgomerie 2003a). Chroma was measured as the proportion of reflectance occurring in the region of interest in relation to the total of the spectrum (300
700 nm) spectrum (Andersson et al. 1998, Doucet and Montgomerie 2003a). Hue was estimated as (lR50), the wavelength at which reflectance is halfway between its minimum JOURNAL OF AVIAN BIOLOGY 37:6 (2006)

(Rmin) and its maximum (Rmax) (Pryke et al. 2001, Bleiweiss 2004). However, these methods to calculate hue and chroma are useful to give a single value of chroma and hue only if the spectrum has one peak, but calculation of these parameters is more complicated when the spectrum considered is bimodal (Doucet and Montgomerie 2003b). Hence, in the case of the yellow breast spectrum of the great tit, which has two peaks, we need to analyse them separately (Doucet and Montgomerie 2003b). Thus, we considered two separated peaks, one for the UV range and the other for the visible one. This is similar to the approach used in other studies, which have either truncated the spectrum in order to study the visible (Grill and Rush 2000, Perrier et al. 2002), UV (Andersson et al. 1998, Doucet 2002, Siitari and Huhta 2002) or both ranges (Doucet and Montgomerie 2003b, Bleiweiss 2004). Nevertheless we have to point out that in the case of the great tit, UV and visible peaks are highly correlated (r2 /0.69; P B/0.001) which means that any change in the visible area affects practically in the same way to the UV-peak and that UV could be a ‘‘by product’’ of carotenoid content in feathers (Mays et al. 2004). Additionally, we used a spectrophotometer Minolta CM-2600d which provides direct values of lightness, chroma, and hue from the visible range. Minolta spectrophotometer collects reflectance spectra by hemispherical directional reflectance, compared to Ocean USB2000 spectrometer which collects reflectance by directional reflectance. Hence, although reflectance of both spectrometers were considerably correlated (r2 /0.64; PB/0.01 N /12), the comparison of the two devices allowed to test for possible differences between the two most common approaches to collect colour variables. Since the yellow breast of the great tit is a lutein-based coloration, the maximum of absorbency at 440
450 nm (range of maximum absorption of lutein; Saino et al. 1999) has also been used to describe plumage coloration (Ninni 2003). For this reason we also measured the repeatability of this value (‘‘lutein peak’’) and compared values obtained from collected feathers and on the bird. For each bird (N /12), we first measured plumage coloration in the field at a standard point in the breast directly on the plumage (Figuerola et al. 1999). The probe of Ocean USB-2000 spectrometer was first placed on this point and subsequently we repeated the measurement at the same point using the Minolta spectrophotometer. Colour samples with Ocean optic were obtained with a fibre optic probe orientated perpendicularly to plumage surface and leaving a distance of 6 mm between the surface of feather and the probe (Keyser and Hill 1999). The size of the measuring spot considered was 54 mm2. We then collected about 15
20 feathers from each bird from the same area where direct JOURNAL OF AVIAN BIOLOGY 37:6 (2006)

colour measurement had been obtained. Once in the laboratory, we disposed 10
12 feathers from each individual by superimposing four layers of three feathers each one (see Fig. 2) on a dark velvet surface (reflectance 0%), trying to imitate the plumage surface of the bird (Bennett et al. 1997). Afterwards we measured the samples with the same fibre optic probe and the same setting as in the field with both the Ocean optics and the Minolta spectrometers. In order to verify the reliability of field and laboratory procedures, first we calculated the repeatability for colour variables for the different methods according to Lessells and Boag (1987), and Harper (1994), from two independent measurements obtained in the field. The second measurement was done without previous knowledge of the first one, two hours after the first measurement was obtained. In the second measurement birds were chosen randomly. The observer was blinded to know which animal was measuring and the spectrum resultant in all measurements. Birds were kept in bags during the period between measurements to avoid that birds were soiled. Repeatability of laboratory measurements was similarly obtained by carrying out two independent measurements of the previously collected feathers, obtained by removing and disposing feathers again before the second measurement was done. The same observer (JQ) did all measurements. In order to establish the comparability of the laboratory method in relation to the plumage obtained by directly measuring from the live bird, we calculated both the repeatability and the correlation between plumage coloration measurements obtained in field and laboratory (averaging the two measurements obtained from each method). To determine the possible effect of the number of feathers on the values obtained from the different plumage colour variables, we measured plumage colour using 2, and systematically adding 3, 4, 5, 7, 10 and 15 feathers from the same individual always measuring in the same point. The procedure was repeated twice and

Fig. 2. Schematic figure of disposition of the collected feather to be measured by spectrometer. The circle represent the area where the measurement was taken.

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we used the mean for analyses. We conducted a Repeated-ANOVA where the number of feathers was the level (dependent variable). We then calculated a Dunnet post-hoc analysis where we used the direct measurement on the bird as control in order to test if there were meaningful differences in colour parameters in relation to the number of measured feathers from the same individual and direct measurements of the bird.

Results Repeatability of colour measurements obtained in the field and in the laboratory Plumage colour measurement obtained directly on the bird in the field proved to be reliable, with lightness, chroma and hue being highly repeatable (Table 1), both for UV and visible ranges, measured with Ocean optics spectrophotometer and also in the visible range measured with Minolta spectrophotometer. Measurement of collected feathers were also highly repeatable in UV range. Measurements on the visible range obtained with Ocean optics and Minolta spectrometers were similarly reliable (Table 1). The repeatability obtained from the comparison of field and laboratory values was also high for lightness but moderate for hue in the UV range and chroma in the UV and visible ranges, as measured with Ocean optics spectrometer. Hue values obtained from plumage (directly measurement) did not differ from that obtained from collected feathers (sign-test t(11) / /0.97; P/0.35), which does not suggest any systematic bias. However this was not the case of chroma (sign-test t(11) /3.73; PB/ 0.01), in which we found that collected feathers provided

a lower measurement of chroma than those measured directly on the bird (visible-chromaCollected feathers: mean9/SD/0.819/0.2; visible-chromaDirect measurement: mean9/SD/0.829/0.1). However, the chroma repeatability between both procedures increased if we used 15 feathers rather than the 12 standard ones used in the other measurements (ri /0.77; PB/0.01 for both UV and visible ranges). Minolta spectrometer provided more consistent results of repeatabilities for hue and chroma in the visible range than Ocean optics when comparing collected feathers and direct measurements on the bird (Table 1). In relation to the peak of lutein, this measurement also gave high values of repeatability both from direct measurements on the bird and from collected feathers, but also when comparing the two procedures (Table 1) in both Ocean Optics and Minolta spectrometers. We found a very high positive and significant correlation between field and laboratory measurements in the UV range for lightness and chroma (lightness: r2 /0.73, P B/0.01; chroma: r2 /0.71, PB/0.01), and a moderate correlation for hue (r2 /0.43, PB/0.05). In the visible range, Ocean optic spectrometer provided similar results than for UV range, a high correlation for lightness and chroma (lightness: r2 /0.81, PB/ 0.01; chroma: r2 /0.71, PB/0.01), but the correlation for hue in the visible was greater than in the UV (r2 /0.60, P B/0.05). The correlation for the lutein peak measured with this spectrometer was moderate but significant (r2 /0.59, P B/0.01). In the case of the Minolta spectrophotometer, repeatabilities between field and laboratory procedures were moderately correlated for all variables (lightness: r2 /0.45, P B/0.05; chroma: r2 / 0.52, PB/0.05; hue r2 /0.67, P B/0.01; lutein peak: r2 / 0.45, PB/0.05).

Table 1. Repeatability in measurements of plumage coloration with a spectrometer from direct measures of bird’s plumage, from collected feathers and comparing both approaches. OCEAN OPTICS UV range

MINOLTA Visible range

Visible range

F

ri

F

ri

F

ri

Directly on plumage Lightness Chroma Hue Lutein peak

67.59 89.04 108.77

0.97*** 0.95*** 0.98***

36.26 36.87 21.24 37.07

0.95*** 0.95*** 0.91*** 0.95***

44.02 22.77 13.70 14.32

0.96*** 0.92*** 0.86*** 0.87***

Collected feathers Lightness Chroma Hue Lutein peak

7.03 39.75 135.96

0.97*** 0.98*** 0.98***

6.73 89.04 29.61 16.13

0.74*** 0.98*** 0.93*** 0.88***

8.83 52.06 22.48 15.20

0.80*** 0.96*** 0.91*** 0.88***

11.62 3.42 4.87

0.84** 0.55* 0.66**

11.14 3.42 7.53 4.26

0.84** 0.55* 0.77** 0.62**

6.33 7.20 9.22 7.11

0.73** 0.76** 0.80** 0.75**

Comparison of both procedures Lightness Chroma Hue Lutein peak *PB/0.05; **PB/0.01; ***PB/0.001.

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JOURNAL OF AVIAN BIOLOGY 37:6 (2006)

Since we had to truncate the spectrum, we additionally tested the ‘‘total lightness’’ (300
700 nm) with the Ocean optics since it is the only measurement which may be done with the whole of spectrum. Results were highly repeatable for all procedures (directly on plumage /0.97; collected feathers /0.78; both procedures/0.83; all P B/0.01) and highly correlated when we used 10
12 feathers (r2 /0.64; PB/0.01).

in Table 2). The Minolta spectrophotometer, however, needed only seven feathers to give comparable measurements between both procedures (Fig. 2j). For the lutein peak both spectrometers gave the same results since no more than four feathers were needed not to find significant differences between collected feathers and direct measurements on the bird (Fig. 2g, k).

The effect of number of feathers used to measure plumage coloration

Discussion

The number of feathers used in plumage colour measurement influenced the value of the different variables defining colour. Lightness dramatically increased when we added more feathers to our sample in all cases (Fig. 3a, d, h). In the UV region, the value of colour stabilised, in relation to that obtained from bird plumage, when we used more than seven feathers, and in the visible region when more than 10, for both spectrometer measurements, as shown by post-hoc analysis (Table 2). Chroma, with the Ocean optic spectrometer, decreased in the UV (Fig. 2b) but increase in the visible region when increasing number of feathers (Fig. 2e). In both cases, chroma began to stabilise from ten to fifteen feathers on, in relation to plumage coloration measured on the bird (Table 2). The Minolta spectrophotometer, however, provided reliable measurements already from 10 feathers on. Hue for both UV and visible range varied as much as lightness, so that this variable stabilised from ten feathers on with Ocean optics spectrometer (Fig. 2c, f; post-hoc analysis

30

20

390

(b)

(c) UV-Hue (nm)

UV-Chroma (%)

20

18

360 345

16

Mean ± 0.95*SE

330

84

40 30 20

560

(e) Vis-Hue (nm)

(d) Vis-chroma (%)

50

375

82

80

(f)

8

Lutein peak (%)

UV-Lightness (%)

(a)

10

Vis-Lightness (%)

Ocean optics

22 40

Measurement of plumage coloration with a spectrometer directly on the bird is a priori the most realistic procedure to determine plumage coloration. However, since spectrometers are sensitive devices, often not fully portable, carrying them at the field is not advisable nor comfortable (Berggren and Merila¨ 2004). A proper alternative is to collect feathers from the colour patch subject to study and to measure colour in the laboratory on a non-reflexive surface (e.g.: black velvet). The method allows a more rapid manipulation of the animal (although pulling out feathers in some delicate species may be not advisable), and also allows the storage of the feathers for subsequent measurements, either of colour or of pigment content (although they have to be properly preserved from light and feather degrading bacteria) (Burtt 1981, Burtt and Ichida 1999). A key assumption to the use of collected feathers is that colour properties obtained from feathers are representative or at least correlated with plumage colour obtained directly on the bird. This can be especially the case when measuring small feathers (e.g. contour

555 550 545

50 40

102

(i)

9

(j)

(k)

100

20 15 10

Lutein peak (%)

60

25

Vis-Hue (º)

(h) Vis-Chroma (%)

Minolta

Vis-Lightness (%)

4

78

30

70

6

2

540 10

(g)

98 96 94

5

3

4

5

7

10

15 Bird

Number of feathers

0

2

3

4

5

7

10

Number of feathers

15 Bird

7 6 5

92

2

8

2

3

4

5

7

10

Number of feathers

15 Bird

2

3

4

5

7

10

15 Bird

Number of feathers

Fig. 3. Effect of number of feathers measured on plumage coloration. All colour parameters are strongly affected by the number of feathers used in the sample. JOURNAL OF AVIAN BIOLOGY 37:6 (2006)

613

Table 2. Dunnet post-hoc analysis of Repeated ANOVA comparing the values of colour parameters obtained with a different increasing number of feathers to that obtained directly on the bird as a control. Using less than 10
15 feathers implies a misrepresentation of real plumage coloration. Lightness

Ocean Optics

Minolta

UV

Visible

Visible

No. of feathers 2 3 4 5 7 10 15

*** *** *** ** 0.15 0.37 0.08

*** *** *** *** * 0.47 0.11

*** *** *** *** *** 0.13 0.37

Chroma 2 3 4 5 7 10 15

*** *** *** *** ** * 0.77

*** *** *** *** ** * 0.83

*** *** *** *** * 0.84 0.98

Hue 2 3 4 5 7 10 15

*** *** *** ** * 0.31 0.52

* ** ** 0.52 0.41 0.84 0.75

*** *** *** * 0.43 0.82 0.85

*** * 0.31 0.98 0.96 0.43 0.61

*** *** 0.35 0.11 0.41 0.88 0.30

Lutein peak 2 3 4 5 7 10 15 *PB/0.05; **PB/0.01; ***PB/0.001.

feathers of Passerines) which are normally not so thick to become opaque, so that plumage colour is normally attained by the superposition of several feathers. Our result that at least 10
15 feathers are needed to obtain maximum reflectance in the contour feather of great tit for both measurements done with different spectrometers, supports this view. Our results stresses the need to use a minimum number of feathers, which may vary from one species to another and from one feather track to another, to attain proper colour reflectance from collected feathers. The repeatability and correlation of plumage colour variables obtained both from collected feathers and directly from plumage on the bird, were high for lightness and hue, which means that measuring colour components of plumage from collected feathers is highly reliable in relation to these parameters. However, repeatability of chroma in the UV and visible range and the correlation between colour obtained from feathers or on the bird, were lower when using the Ocean optics spectrometer. However, this was an effect of number of 614

feathers used, since when using/15 collected feathers we obtained comparable measurements when comparing to the direct measurement on the bird plumage. We should also stress that the repeatability of some measurements, as lightness in the visible range measured with Ocean optics (ri /74%) or the peak of lutein, could probably be increased by increasing the number of measurements from each sample and averaging them (Harper 1994, Senar 1999). That is in fact what has already been done in several studies (Bennett et al. 1997, Keyser and Hill 1999, Doucet 2002, Siefferman and Hill 2004, Alonso-Alvarez et al. 2004). Our results also demonstrated that the addition of more feathers to the sample strongly affects colour values from collected feathers. In general, and in order to make comparable the colour values obtained from collected feathers to that from a direct measurement on the bird, a minimum of 15 feathers are needed when using Ocean optics spectrophotometer (directional reflectance), and a minimum of 10 for when using Minolta device (hemispherical directional reflectance). In the case of the lutein peak, four feathers are enough to make a good reading of colour properties from plucked feathers. The change in colour by adding more feathers is probably related to the fact that the feather thickness affects the readings since a thicker surface of feathers is more likely to reflect less light because a part is lost. The use of a minimum number of feathers to provide a good reading raises the question about if sampling 10
15 feathers could inflict any harm to the birds. Thus, researchers should take into account this topic, particularly in long-term studies which implies to pull out feather several times so that the statistical benefits of more feather to the sample should not outweigh the harm to the survival of the birds. Nevertheless, we should stress here that in the case of a great tit (and similar sized birds), 15 feathers is a minimum portion of the feathers in the track and that collecting these feathers do not seems to have harmed the birds in any way. We should also stress that the larger and the thicker the feather is, a more reduced number of feathers may be needed. Summarising, spectrometers provide reliable measurements of plumage coloration, both when obtained directly on the animal or from collected feathers, and both techniques are nicely correlated. However, data shows that when measuring colour from collected feathers we must use an adequate number of feather to get reliable data. In general, and for plumage colour measurements from contour feathers of small passerines, a minimum of 10
15 feathers should be used. Acknowledgements
We thank Lluı¨sa Arroyo and Francesc Sarda´ for their assistance in the field. Hermanitas de la Asuncio´n kindly allowed the access to their property. The authors also thank Susana I. Santos for technical advice with spectrum and to Esther del Val and two anonymous referees for useful comments to the manuscript. Authors had the permission JOURNAL OF AVIAN BIOLOGY 37:6 (2006)

to make this study from Environmental Department of the Regional Catalonian Government (permission number to JQ SF/125). This work was funded by Research Project BOS 2003-09589 to JCS and FPI Grant FP2000-6439 to JQ from the Spanish Research Council, Ministero de Ciencia y Tecnologı´a.

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JOURNAL OF AVIAN BIOLOGY 37:6 (2006)