Ornithol Sci 5: 133–137 (2006)

SHORT COMMUNICATION

Molecular sexing and sexual difference in carpal spur lengthof the Gray-headed Lapwing Vanellus cinereus(Charadriidae)
ORNITHOLOGICALSCIENCE© The Ornithological Societyof Japan 2006

Hideya WAKISAKA1,*, Munetaka NAKAGAWA2, Keiko WAKISAKA1 andMasanobu ITOH1,3,#1

Dept. Applied Biology, Faculty of Textile Sciences, Kyoto Institute of Technology, Matsugasaki,
Sakyo-ku, Kyoto 606–8585, Japan2
Wild Bird Society of Ogura, Minami-Kakiuchi 13, Nishi-Tono, Joyo, Kyoto 610–0111, Japan3
Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku,Kyoto 606–8585, Japan

Carpal spurs are projecting bony cores covered
with a keratinous outer layer on the forward edge ofthe bird wing near the carpal joint. Prominent carpal
spurs are found in all species of screamers (Anhimidae) and in some species of geese (Anseridae), jacanas (Jacanidae), and plovers (Charadriidae); they
are usually well developed in both sexes, however
males usually have slightly longer spurs than do females (Rand 1954; Prater et al. 1977). These projections of the carpalia are, anatomically, processes ofmetacarpal I, the original function of which was as an
attachment of the extensor muscles (Rand 1954).
There have been few studies focusing on the functionof wing spurs, and their biological significance remains to be elucidated.
In the Spur-winged Plover Vanellus spinosus,
which has one of largest carpal spurs for a bird of its
size, the spurs have been considered to function as aform of armature (Cramp et al. 1983), because during
their threat display (towards other plovers or intruders) their spurs are brandished demonstratively. Incontrast, both female and male of the Northern Jacana, Jacana spinosa, are reported to spread theirwings and attempt to strike each other with theircarpal spurs during courtship (Rand 1954).
The Gray-headed Lapwing, V. cinereus, is one of
the members of the Charadriiformes that has carpalspurs. Because the nests of this species are constructed on the ground of open land, they are particularly suitable for observation and capture to investi-gate functional aspects of carpal spurs. Information
concerning the species is, however, somewhat contradictory, with some authors finding no conspicuous
morphological differences between the sexes (Okugawa et al. 1970; Prater et al. 1977), whereas others
have referred to males having longer carpal spurs
than females (Kobayashi 1956; Sakane 1957). An initial goal of this study was to investigate the spurs of
the Gray-headed Lapwing and to confirm whether
sexual dimorphism in spur size occurs in this species
or not.
Molecular typing of the Chromo-Helicase-DNAbinding protein (CHD) genes has recently beendeveloped as a reliable sexing system for many
avian species (Ellegren 1996; Griffiths et al. 1996;
Fridolfsson et al. 1998; Kikuchi & Ishii 2002; Mizutaet al. 2003; Seki 2003). This technique has also
proven successful among the Charadriidae for sexing
Kentish Plover Charadrius alexandrinus (Blomqvistet al. 2002; Szekely et al. 2004). As a means of investigating the extent of sexual dimorphism in the Grayheaded Lapwing, we first attempted to sex (molecularly) wild-caught birds, then compared the externalmeasurements of females and males.

MATERIALS AND METHODS1)

Birds
Adult Gray-headed Lapwings were captured singlyat their nests by means of a noose trap (under permission of the Ministry of the Environment, Japan) on
the Ogura reclaimed land, Kyoto, Japan (34.9°N,135.8°E) in spring (April and May) 2003 and 2004.
All nests had been constructed in rice fields prior tothose fields being flooded. After measuring their ex-(Received 31 August 2005; Accepted 3 February 2006)#Corresponding author, E-mail: mitoh@kit.ac.jp* Present address: Himeji Ise Shizen-no-sato Environmental Learning Center, Section of Sanctuary, Wild Bird Society of Japan,Odutsumi 615, Hayashida, Himeji, 679–4231, Japan133

H. WAKISAKA et al.

ternal body features and taking blood samples, eachbird was marked with a numbered metal ring provided by the Ministry of the Environment and released immediately. Twenty six individuals from 22
nests were studied.
2)

Genomic DNA and PCR
Small blood samples (approximately 50 m l) wereobtained by brachial vein puncture, following Kajita(1999), and stored in 99% ethanol at room temperature until use. Genomic DNA was extracted using acommercial kit (AquaPure Genomic DNA Isolation
Kit, Bio Rad). Each PCR reaction was carried out ina 50 m l mixture containing 0.25 units of DNA polymerase (KOD Dash, TOYOBO), 2.5 mM each ofdNTPs, and 10 mM each of the primers 2550F (5GTTACTGATTCGTCTACGAGA-3) and 2718R(5- ATTGAAATGATCCAGTGCTTG-3) for amplifying the CHD genes (Fridolfsson & Ellegren 1999)
using a thermal cycler (The Mini Cycler PTC-15016, MJ Research). The thermal profile was as follows: initial denaturing at 94°C for 2 min followed bya touch-down scheme where the annealing temperature was lowered by 1°C per cycle from 60°C to50°C, and 30 additional cycles including denaturing
at 94°C for 30 sec, annealing at 50°C for 30 sec, andextension at 74°C for 30 sec. A final extension step at74°C for 4.5 min was added. PCR products were
electrophoresed on a 3% agarose gel at 100 V for
30 min and revealed with ethidium bromide stain.

Fig. 1. Measurments. a: wattle length, b: wattle width, c:
CSL (carpal spur length), d: breast-band depth, and e: tailband depth.

for discriminating between the sexes, regression
analysis was performed using the VBA Analysis toolof Microsoft Excel 2001.

RESULTS1)

Molecular sexing of the Gray-headed Lapwing
Genomic DNA was successfully extracted from 26
wild caught Gray-headed Lapwings. To determine
each individual’s gender we performed PCR to amplify a portion of the CHD genes (Fig. 2). All birds
showed a PCR product of about 600 bp, which is expected if the template is the CHD-Z gene on the Z
chromosome. Some birds showed an additional
450 bp PCR product and others did not, suggestingthat the former were females (ZW) and the latter
were males (ZZ), because the 450 bp product is expected if the template is the CHD-W gene on the W
chromosome. This is consistent with the results forthe chicken, Gallus domesticus, of known sex, themales of which showed only a 600 bp band whereas
the females showed two bands of 600 bp and 450 bp.
On this basis, we identified 12 individual Grayheaded Lapwings as females and 14 as males.

3)

External morphological features
According to Svensson (1970), we measured thefollowing characters of each individual using calipers
and rulers: total length, wing length (unflattened),
total head length, total bill length, bill depth (at proximal edge of nostrils), bill width (BW; at proximaledge of nostrils), tarsus length, and tail length. The
measurements for carpal spur length (CSL; length ofthe longer one), breast-band depth, tail-band depth,
wattle length (length of the longer one), and wattle
width (width of the wider one) are shown in Fig. 1.
Each bird was also weighed using a balance.
4)

Data analysis
The differences between measurements of males
and females were tested statistically using theWilcoxon two-sample test. Since we performed 14
tests for the external features, the critical value for
the Bonferroni multi-test correction was 0.05/14
0.00357. To derive the Fisher’s discriminant function2) Comparison of external features between females and males
Following the results of molecular sexing using theCHD genes, we compared the 14 external measure134

Molecular sexing and carpal spur of V. cinereus

Fig. 2. Molecular sexing of birds using PCR of the Chromo-Helicase-DNA-binding protein (CHD) genes forGray-headed Lapwings. Pairs are numbered from I to IV. Female and male chickens, Gallus domesticus, were used
as controls. F: female, M: male.
Table 1.

Mean values of morphological measurements of the Gray-headed Lapwing.
CharactersFemale

Male

P-value

Total length
Wing length
Total head length
Total bill length
Bill depthBill widthTarsus length
Tail length
Carpal spur length
Breast-band depth
Tail-band depth
Wattle length
Wattle width
Body weight336.312.1 (12)235.54.6 (12)73.301.97 (12)41.373.00 (12)10.150.39 (12)8.500.49 (12)76.263.45 (12)109.82.8 (11)3.690.54 (12)11.63.7 ( 8)28.13.2 ( 8)5.170.66 (12)5.031.03 (12)266.619.7 (10)341.712.1 (14)236.97.5 (14)74.291.77 (13)42.022.91 (14)10.430.56 (14)8.790.43 (14)76.902.70 (14)109.43.7 (14)5.081.35 (14)12.64.2 ( 8)29.53.6 ( 8)5.711.36 (14)5.621.12 (14)280.115.9 (13)0.29170.66200.22100.48750.26170.13580.35450.33800.0006*
0.58490.59050.25780.22680.1629The units of measurements, meanstandard deviation, are g for body weight and mm for all others. The numbers of individuals
are in parentheses. P values are from Wilcoxon two-sample tests. Asterisk indicates P0.001.

ments, including CSL, of males and females (Table1). Mean values of all measurements were found tobe larger in males than in females, with only one exception, which was tail length—females had longer
tails. Wilcoxon two-sample tests indicated that thegender difference was significant only for CSL(P0.0006, which was significant even after the Bonferroni correction), but not in other characters(P0.10). However, the range of values overlapped
considerably, even for CSL, which ranged from
3.0 mm to 4.9 mm for females and from 3.8 mm to9.0 mm for males. Furthermore, the spur length of themale was not always longer than that of his probable
mate; in two pairs the female’s spurs were longer
than her mate’s (Fig. 3a).
By taking CSL as the explanatory variable after
standardizing by BW, a discriminant function,D4.293(CSL/BW)2.117, was derived. This func-tion correctly categorized the sex of 81% (21/26) ofthe birds (Fig. 3b). None of the attempts at improving
the reliability were successful; for example, adding
other parameters as explanatory variables (data notshown).

DISCUSSION
Among the 14 external features of the Gray-headed
Lapwing that were measured, only CSL was shownto differ significantly between the sexes—spurs werelonger in males than in females. Sexing, by means of
the molecular typing of the CHD genes, was unambiguous. The sexual difference in CSL observed here
was not an effect of differences in body mass between males and females, but a result of disproportionate growth of the carpal spurs. Our results are
consistent with the previous reports of sexual dimor135

H. WAKISAKA et al.

cating the importance of carpal spur size for mating
success. Gray-headed Lapwings are, however, known
to make a threat posture, during which they extend
their wings horizontally, when intruders approach
their nests. Many such threat displays were made towards us while we were setting the traps in this study(data not shown), and it appeared as if they were attempting to display their carpal spurs to us. Thesevarious results taken together indicate that the Grayheaded Lapwing uses its wing spurs not in mating behavior, but as weapons, in much the same way as theSpur-winged Plover (Cramp et al. 1983). Even if thisis the case, however, several questions concerning thefunction of the wing spurs remain unanswered; inparticular the reason for the sexual difference in CSL.
Accurate molecular sexing, by means of the CHD
genes will facilitate further investigation.
ACKNOWLEDGMENTS
Fig. 3. CSL of Gray-headed Lapwing. (a) CSL (carpal spurlength) of individuals in pairs. Birds and pairs are numbered asshown in Fig. 2. White bars indicate females, and black bars
males. (b) Distribution of CSL with standardizing by BW (billwidth). The broken line is based on the discriminant function,D4.293 (CSL/BW)2.117, and indicates the demarcation
between females and males (D0). This is almost equivalentto using the value of CSL/BW of 0.5 (2.117/4.293) or larger as
an indicator of maleness. Open circle: female; closed circle:male.

We thank M. Kajita, H. Sugawa, Y. Ezaki, and H. Arima for
their technical advice and encouragement throughout thiswork. We thank I. A. Boussy and N. Yoshida for informative
suggestions on data analysis and helpful discussion. Our
thanks are also due to M. Brazil for careful reading the manuscript, as well as to anonymous referees for their valuablecomments.

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Molecular sexing and carpal spur of V. cinereus

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