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

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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 combina- tion. 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 protuber- ances (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.
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