An Essential Prerequisite for Studies of Social Organization of a Species Is the Accurate Determination of an Individual's Sex

山階鳥研報(J.Yamashina Inst.Ornithol.),27:78-88,1995

Sex Determination Based on Cloacal Protuberances in the Japanese Accentor Prunella rubida

Masahiko Nakamura * and Yoshiyuki Matsuzaki * 1

Abstract External morphological measurements and the characteristics of cloacal protuberances were investigated to find a useful index of sexing the Japanese Accentor Prunella rubida. From 20 May to 15 June in 1989-1990 and 1992, morphological variations in eight characters were measured in known-sexed 58 males and 36 females inhabiting on the top of Mt. Norikura, Japan. The males were significantly heavier and had longer wings and tails than females, but these measurements could not be used as indexes for sexing because there was still considerable overlap. No sex-related difference in size was evident in the five skeletal elements monitored (tarsus, three bill dimensions and head length). The sharpness of the cloacal protuberance of each sex proved to be the most useful index to identify the sex of individuals. The males developed a nodular cloacal protuberance formed by the growth of the distal ends of sperm-filled ductus deferens, while the female's cloaca protruded from its original position to form a cylindrical protuberance. Because the cloacal protuberance of each sex developed only during the breeding season, its use as a characteristic for sex is confined to the breeding season. Male Japanese Accentors had a relatively large cloacal protuberance and testes compared with other passerines. It seems that these large reproductive organs are linked to the intense sperm competition predicted from this species' polyandrous or polygynandrous mating combination. Key words: Cloacal protuberance, Japanese Accentor, Morphological measurement, Sex determination.

Introduction

An essential prerequisite for studies of social organization of a species is the accurate determination of an individual's sex. The Japanese Accentor Prunella rubida, endemic to Japan and southern Kuriles, is a common passerine in the Japan Alps up to timber line. However, the identification of sex is difficult because this species is sexually monomorphic in plumage, and because both males and females sing (Y. Matsuzaki unpubl. data). For passerine species with no readily visible sex-dependent indicators, sex is often determined from morphological measurements (Abe 1984, Ueda 1987, Nakamura et al. 1989). It is also possible to determine sex according to the external characteristics of cloacal protuberances (Drost 1938, Mason 1938, McCabe 1943, Wolfson 1952). In most (if not all) passerine males, the seasonal growth of the distal ends of sperm-filled ductus deferens, the seminal glomera, forces the posterior wall of the cloaca to form a nodular cloacal protuberance (Wolfson 1952, 1954, Salt 1954). The present paper forms part of a 3-year study of the social organization of the

Received 22 April 1995, Revised 15 July 1995, Accepted 19 July 1995. * Department of Biology , Joetsu University of Education, 1 Yamayashiki-machi, Joetsu-shi, Niigata 943, Japan *1 Present address: Imoi Primary School , 753 Iriyama, Nagano-shi, Nagano 380, Japan

78 Sex determination in the Japanese Acceptor 79

Japanese Accentor. Here we investigate eight external morphological measurements and the external reproductive organ of this species to determine a sexing index. A congeneric species, the Alpine Accentor P. collaris has the largest male cloacal protuberance for its body-size of any known bird (Nakamura 1990, Birkhead et al. 1993) and male Dunnocks P. modularis also have large cloacal protuberances (Birkhead et al. 1991), but the protuberance of the Japanese Accentor has not been reported before. To examine whether it is a useful index for sexing, we describe the features and size of cloacal protuberances of each sex.

Study Area and Methods

The present study was conducted from May through September in 1989-1990 and l992 on the 15 ha summit of Mt. Norikura, central Honshu, Japan (36°06′N, 137°33′E, 2,664-2,817 m alt.). Adult birds were captured in mist nets between dawn and 0900, colour banded and then released at the study site. During the banding process, eight measurements were recorded for birds of known sex: (1) body weight, weighed to the nearest 0.1 g using a digital scale (TANITA, HANDYMINI 1476); (2) wing length, the maximum distance from the carpal joint to the tip of the longest primary in the flattened and straightened right wing; (3) tail length; (4) tarsus length; (5) bill length; (6) bill depth; (7) bill width; (8) head length, the maximum distance from the bill tip to the back of the skull. We also measured three cloacal dimensions (length, width and height, see Figs. 3a and 5a) in each sex (males, measured on 106 occasions, n=56; females, 57 occasions, n=35). All linear measurements were taken to the nearest 0.1 mm with vernier callipers. Sex was determined by the two independent methods of dissection and copulatory behaviour. We categorized individuals mounted at mating as males and birds who presenting pink cloacal lips during pre-copulatory displays as females (see Result section for details). Because incubation was performed only by birds that adopted the cloaca- presenting behaviours, the sex of incubating individuals was determined as female. Throughout the three years of sampling, 58 males and 38 females were captured a

Table 1. Means±S.E. and ranges of eight measurements of male and female Japanese Accentors (body

weight in grams, other measurements in mm). 80 M.Nakamura and Y. Matsuzaki

Fig. 1. Photographs of the fully developed cloacal protuberance of a male Japanese Accentor . (a) lateral view, (b) dorsal view and (c) phallus (arrow) extruded from cloacal protuberance (photographed respectively on 22, 25 and 12 June in 1992). Sex determination in the Japanese Accentor 81

Fig.2. Spermatozoa in the ductus deferens at the cloacal protuberance. Scale bar=30μm. total of 110 and 58 times, respectively. In order to exclude any bias from seasonal fluctuations in the eight measurements, data collection was restricted within 20 May to 15 June in each year. This period corresponded to the pre-breeding season in this population. There was no significant year-to-year variation in the eight measurements (Kruskal-Wallis test,P<0.05 in allcases), so the data from allthe years were combined. Results are presentedas means with S.E. and the statisticalanalyses are done by two-tailedStudent's t-tests. Two males and a single female were collected; one male was killed by the Stoat Mustela erminea on 20 August in 1990, and another male and a female died from hitting the windows of mountain cottages on 22 June in 1992 and on 17 June in 1992, respectively. These birds were dissected to examine their respective reproductive tracts. For the males, both testes removed and separately weighted to the nearest 0.1 g using the digital scale and the length and breadth were taken to the nearest 0.1 mm with vernier callipers. The cloacal protuberance of the specimen collected on 22 June in 1992 was removed and embedded in paraffin blocks. Serial cross sections (4μm thick) were stained with haematoxylin and examined under a microscope. For the female, the body cavity was opened and the development of ovary was confirmed.

Results 1. External morphological measurements Males had significantly greater body masses and longer wings and tails than did females, but the range of each measurement showed considerable overlap (Table 1). No sex-related difference in size was evident in the five skeletal elements monitored (tarsus length, three bill dimensions and head length).

2. Male's cloacal protuberance Male Japanese Accentors had a bulbous cloacal protuberance during the breeding 82 M. Nakamura and Y. Matsuzaki season (Fig. 1). The shape of the protuberance varied little between males . The fully developed cloacal protuberance was mostly bare except for a ring of feathers around the vent (Fig. 1) and the dissection of a male collected on 22 June in 1992 revealed it to be formed by two large pear-shaped bodies stuffed with the coiled seminal glomera . Micro- scopic sections of seminal glomera showed that they contained very large numbers of spermatozoa (Fig. 2). By gentle lateral squeezing, the protuberance everted to reveal a large, red, phallus-like structure, terminating in a small papilla (Fig . 1c), from which we could extrude a droplet of semen. The average dimensions of the fully developed protuberance were: length, 8.8±0.1mm; width, 9.2±0.1mm; height, 9.6±0.2 mm (n= 38 males).

Fig. 3. Seasonal changes in the size of the male protuberance throughout the breeding season. a, b and c show the seasonal change in the size of the three labelled dimensions of the cloacal region . Data (n=106) were pooled for the 3 years. Solid lines connect the data points of the same individual. Sex determination in the Japanese Acceptor 83

Fig. 4. Photograph of the cloacal protuberance of a female Japanese Accentor (photographed on 28 June in 1990). Vent is surrounded by tuft of feathers; head to right.

The size of the male's cloacal protuberance changed seasonally (Fig. 3). The males cloacal region swelled gradually, from the end of May to mid June, and the vent protruded about 7 mm from its original position by mid-June (Fig. 3). From 10 to 20 June, the coiled ducts of the seminal glomera could be seen through the skin of the protuberance in three out of 11 individuals, but the glomera were no longer visible by the end of June. The peak condition of the protuberance was maintained for about 50 days, from the middle of June to late in July (Fig. 3). Once developed, the protuberance showed no changes in size. Toward the end of August, the protuberance regressed and disappeared (Fig. 3). During the 3-year study, the earliest laying date was 12 June in 1989 and the latest laying date was 8 August in 1989. Therefore, the development period of the male's protuberance overlapped completely with the breeding season.

3. Testes mass

The dimensions of testes of a specimen collected on 22 June in 1992 were: right testis

(length×breadth), 9.8×9.1mm, 0.3 g(wet mass); left, 11.2×7.5 mm,0.4 g. The dimensions of testes collected on 20 August 1990 were: right, 2.1×2.3 mm,0.1 g; left, 2.2×3.1 mm,0.1 g. The former seemed to be in reproductive condition and the dimensions of the

cloacal protuberance were: 7.8×8.6×9.2 mm (length×width×height). A few of the

primaries of the latter were moulted and the dimensions of the protuberance were: 5.3× 4.7×3.8 mm.

4. Female's cloacal protuberance The female's cloaca did not exhibit as a clear protuberance as the males, but it showed some swelling as a cylinder and was elevated from the body wall (Fig. 4). The ovary of a female (collected on 17 June in 1992) was developed, but the vent protruded only 2.1 mm from its original position. The females formed the cloacal protuberance from the beginning of June, regressing toward mid-August (Fig. 5). The development period of the 84 M. Nakamura and Y. Matsuzaki

Fig. 5. Seasonal changes in the size of the female protuberance throughout the breeding season. a, b and c show the seasonal change in the size of the three labelled dimensions of the cloacal region. Data (n=57) were pooled for the 3 years. Solid lines connect the data points of the same individual. female's protuberance overlapped with the breeding season. Female Alpine Accentors form a cylindrical cloacal protuberance whose lips swell and turn scarlet (Nakamura 1990). The cloacal lips remain swollen for about a month until incubation begins. The development period of the female's cloacal protuberance corresponds to the copulation period of this species because the female exposes the scarlet lips toward the male, thereby soliciting mountings, and the male copulates only during the development period (Nakamura 1990). Female Japanese Accentors also exposed the cloacal protuberance in order to solicit copulations during the pre-copulatory display but the cloacal lips turned pink only during the display. The scarlet cloacal lips of female Sex determination in the Japanese Accentor 85

Alpine Accentors can be seen even when the birds are agitated by handling, but not visible in female Japanese Accentors. In the Dunnock, during the female's pre-copulatory display in which her cloaca is exposed, the male pecks her cloaca before he copulates to remove sperm from other males (Davies 1983). During the pecking by the male, the female's cloaca becomes pink and distended (Harrison & Binfield 1967, Sanderson 1968, Davies 1983). However, the cloaca-pecking behaviour was never observed in male Japanese Accentors and the female's cloacal lips turned pink without the male pecking.

Discussion Because male Dunnocks are heavier and have longer wings and tarsi than females, these measurements are used as an index for sexing (Birkhead 1981, Davies 1992). Our biometric data indicate that male Japanese Accentors also display greater body masses and longer wings, but a considerable overlap in values was evident (Table 1). Thus, it is impossible to say with any degree of certainty that a bird belongs to one or other sex based on morphological measurements alone. Measurements analysed by linear discriminant analysis would be likely to provide a more reliable sex classification, and be more statistically accurate, than if univariate methods were used. However, a more simple and reliable criterion of identifying the sex of the Japanese Accentor is the external characteristics of cloacal protuberances. Ishizawa (1961) collected six male Japanese Accentors and one female in the Japan Alps in summer, but he did not refer to the cloacal protuberance. The present study shows that male Japanese Accentors possess a definite bulbous cloacal protuberance. The sharpness and size of the male's cloacal protuberance were quite distinct from those of female's. Therefore, the cloacal protuberance can be used to give an indication of the sex in the Japanese Accentor, even by unskilled researchers or technicians. The cloacal protuberance of each sex persisted throughout the breeding season, after which it regressed and disappeared (Figs. 3 & 5). Its use as a diagnostic characteristic for sexing is therefore confined to the breeding season. To determine whether the male Japanese Accentor's cloacal protuberance was large in relation to its body size, we made comparisons with the other species for which data exist. Interspecific comparisons were made by expressing the volume index (length × width × height mm) of the cloacal protuberance as a proportion of body weight (gram).

The volume index of the fully developed protuberance of male Japanese Accentors is 39.9

(8.8×9.2×9.6/19.5). Compared to the same indexes for eight passerines listed in Birkhead et al. (1991) and for the Superb Fairy-wrens Malurus cyaneus (Mulder & Cockburn 1993), the cloacal protuberance of the Japanese Accentor is among the four largest: Alpine Accentor: 55.7, Superb Fairy-wrens: 26.7, Dunnock: 21.7. We also compared the Japanese Accentor's combined testis weight (0.7 g or 3.59% of adult male weight) with that predicted by Moller's (1991) relationship for a range of bird species. The observed testes mass was 2.3 times (0.31 g) larger than predicted for body mass. Testis weight of the Japanese Accentor was similar to that of the Dunnock (0.688 g or 86 M. Nakamura and Y. Matsuzaki

3.36% of adult male weight, Birkhead et al. 1991). The Alpine Accentor has even larger testes: their combined weight was 3.6 g or 7.7% of male weight and the testes mass was 6.5 times (0.55 g) larger than predicted for body mass (Nakamura 1990). The results presented here show that the Japanese Accentor, like the Alpine Accentor and the Dunnock, has a relatively large cloacal protuberance and testes compared with other passerines. The cloacal protuberance is a site for sperm storage and maturation before ejaculation (Lake 1981). Sperm number per ejaculate increases with testes weight (Moller 1988) and the number of sperm stored in the cloacal protuberance are positively correlated with its size (Birkhead et al. 1993). Male Japanese Accentors are therefore morphologically adapted for high levels of sperm production. Birkhead et al. (1991) and Moller (1991) predicted that when sperm competition is intense, as measured by male copulation frequency and/or mating system, males will have relatively large protuberances and testes mass. Briskie (1993) compared the reproductive anatomy of three polygynandrous mating species (the Dunnock, the Alpine Accentor and the Smith's Longspur Calcarius pictus) with other mating ones, and he demonstrated that they have enlarged testes and cloacal protuberances. Matsuzaki (1991) has shown that most female Japanese Accentors are associated with two males in either a polyandrous or polygynandrous mating combination. Although the detail in which they might copulate frequently remains unclear because they usually copulate in dense Pinus pumila scrubs, the female actively exposes the pink protuberance toward several different males on the ground in scrub, while males copulate with several females in rapid succession (Matsuzaki 1991). Thus, sperm competition seems to be an important component of the Japanese Accentor mating system and is reflected by the fact that testes are much larger than predicted by body weight. It seems likely that the male's cloacal protuberance has also evolved in response to sperm competition, presumably as a means of facilitating multiple mating, because this would demand larger sperm reserves and the transfer of large ejaculates.

Acknowledgements

We are grateful to Professor Toru Nakamura for his kind advice and encouragement. Members of Laboratory of Animal Ecology of Joetsu University of Education, Akira Narita, Hiroaki Ootaka, Takashi Haga, Masahiko Yamamoto, Satoko Fujii and Miyako Yamada, helped in the field. We thank Satoshi Murayama for technical assistance. Thanks are also due to two reviewers, Drs. J. J. Thompson and Nariko Oka for their helpful suggestions.

References Abe, N. 1984. The wing length as a sexing criterion of Cettia diphone cantans in autumn routine ringing. J. Yamashina Inst. Ornithol. 16: 151-158. (in Japanese with English abstract). Birkhead, M. E. 1981. The social behaviour of the Dunnock Prunella modularis. Ibis 123: 75-84. Birkhead, T. R., Hatchwell, B. J. & Davies, N. B. 1991. Sperm competition and the reproductive organs of the male and female Dunnock Prunella modularis. Ibis 133: 306-311. Sex determination in the Japanese Accentor 87

Birkhead, T. R., Briskie, J. V. & Moller, A. P. 1993. Male sperm reserves and copulation frequency in birds. Behav. Ecol. Sociobiol. 32: 85-93. Briskie, J. V. 1993. Anatomical adaptations to sperm competition in Smith's Longspurs and other polygynandrous passerines. Auk 110: 875-888. Davies, N. B. 1983. Polyandry, cloaca-pecking and sperm competition in Dunnocks. Nature 302: 334-336. Davies, N. B. 1992. Dunnock Behaviour and Social Evolution. pp. 74-76. Oxford University Press, Oxford. Drost, R. 1938. Geschlechtsbestimmung lebender Vogel nach der Form der Kloakengegend. Der Vogelzug 9: 102-105. Harrison, C. J. O. & Binfield, F. G. 1967. Cloaca-pecking and copulation in the Dunnock. Bird Study 14: 192- 193. Ishizawa, J. 1961. Measurements of the testes and ovaries of birds, collected in Japan Alps range in summer. Tori 16: 355-359. (in Japanese with English summary). Lake, P. E. 1981. Male genital organs. In: Form and Function in Birds [eds. King, A. S. and J. McLelland], Vol. 2, pp. 1-61. Academic Press, New York. Mason, E. A. 1938. Determining sex in breeding birds. Bird-Banding 9: 46-48. Matsuzaki, Y. 1991. Breeding biology and mating system of the Japanese Accentor Prunella rubida at Mt. Norikura. Master's thesis of Joetsu University of Education. (in Japanese). McCabe, T. T. 1943. An aspect of collectors' technique. Auk 60: 550-558. Moller, A. P. 1988. Testes size, ejaculate quality and sperm competition in birds. Biol. J. Linn. Soc. 33: 273-283. Moller, A. P. 1991. Sperm competition, sperm depletion, parental care, and relative testis size in birds. Am. Nat. 137: 882-906. Mulder, R. A. & Cockburn, A. 1993. Sperm competition and the reproductive anatomy of male Superb Fairy-wrens. Auk 110: 588-593. Nakamura, K., Sato, F., Sugimori, F. & Imamura, T. 1989. Sex discrimination based on external morphological measurements in Brown-eared Bulbul Hypsipetes amaurotis. J. Yamashina Inst. Ornithol. 21: 253-264. (in Japanese with English abstract). Nakamura, M. 1990. Cloacal protuberance and copulatory behavior of the Alpine Accentor (Prunella collaris). Auk 107: 284-295. Salt, W. R. 1954. The structure of the cloacal protuberance of the Vesper Sparrow (Pooecetes gramineus) and certain other passerine birds. Auk 71: 64-73. Sanderson, R. F. 1968. Cloaca pecking in the Dunnock. Bird Study 15: 213. Ueda, K. 1987. Sexing of the Fan-tailed Warbler Cisticolajuncidis by wing length. Bull. Jap. Bird Banding Assoc. 2: 35-40. (in Japanese with English summary). Wolfson, A. 1952. The cloacal protuberance-a means for determining breeding condition in live male passerines. Bird-Banding 23: 159-165. Wolfson, A. 1954. Notes on the cloacal protuberance, seminal vesicles, and a possible copulatory organ in male passerine birds. Bull. Chicago Acad. Sci. 10: 1-23.

総排泄腔突起によるカヤクグリの性判定

1.カヤクグリの性判定の有効な指標を見いだすため,複数部位の外部形態測定値と総排泄腔突起の形態を調べた。 2.1989-1990年と1992年の5月20日から6月15日の期間,乗鞍岳山頂部において,すでに性のわかっている雄58個体と雌36個体について8部位の形態を測定した。 3.雄は雌に比べ体重は重く,翼長と尾長は長かったが,いずれもデータの分布にかなりの重複があるため,性判定の指標にはならなかった。5箇所の骨格部位(足付蹠,嘴峰の3部位および全頭長)には雌雄間に

有意な差は認められなかった。 4.雌雄の総排泄腔突起の形態の違いが最も有効な性判定の指標であることがわかった。雄は繁殖期に 88 M. Nakamura and Y. Matsuzaki

輸精管の末端の肥大によるこぶ状の総排泄腔突起を形成し,雌は円柱状の突起を形成した。 5.雌雄の総排泄腔突起は繁殖期のみ発達していたので,総排泄腔突起による性判定は繁殖期に限定される。 6.カヤクグリの雄は,スズメ目の他種に比べ相対的に大きな総排泄腔突起と精巣をもっていた。雄の大きな生殖器官は,本種の一妻多夫あるいは多夫多妻の配偶関係から予想される強い精子競争に起因すると考えた。

中村雅彦:上越教育大学自然系理科生物学教室〒943新潟県上越市山屋敷町1番地松崎善幸:長野市立芋井小学校〒753長野県長野市入山753番地