Behavioral, hormonal, and morphological responses of free-living male pied flycatchers to estradiol treatment of their mates

HORMONES AND BEHAVIOR 25,

38-56 (1991)

Behavioral, Hormonal, and Morphological Responses of Free-Living Male Pied Flycatchers to Estradiol Treatment of Their Mates BENGT SILVERIN Department

of Zoology,

University

of Giiteborg, Sweden

Box 250 59, S-400 31 Gateborg,

A field experiment was performed implanting female pied flycatchers (Ficedula hypoleuca) during the nest-building period with silastic tubes containing 17pestradiol. Control females were given empty silastic tubes. The pied flycatcher is a polygamous and polyterritorial bird species. During two successive breeding seasons effects on reproductive behavior and the reproductive systems of males were studied. Reproductive successwas not affected by the treatment. Estradiol treatment of females caused marked changes in the behavior of males, increasing aggressive and mate-guarding behavior. The results of the present study also support the idea that cuckoldry is a problem for the territory holder. Fewer males paired with estradiol-treated females established secondary territories, and significantly more of the polyterritorial males from the experimental population showed up in the home-territory when a simulated territorial intrusion was performed there. Control males were much more successfulin getting a second female to breed. Spermatogenesis and hormonal cycles (DHT, LH, and testosterone) in males were not prolonged as a result of estradiol treatment of the females. o MI Academic Press, Inc.

INTRODUCTION Behavioral interactions between individuals are important factors in the synchronization of behavioral and physiological breeding activities between a male and a female within a pair. Experimental prolongation of the sexual behavior of free-living female birds dramatically affects gonadal and endocrine systems of their mates in white-crowned sparrows (Zonotrichiu leucophrys) (Moore, 1982) and song sparrows (Melospiza melodiu) (Runefeldt and Wingfeldt, 1985). The presence of a female also affects the time course of testicular and endocrine cycles in caged male willow tits (Parus montanus) (Westin, 1989) white-crowned sparrows (Moore, 1983), starlings (Sturnus vulgaris) (Schwab and Lott, 1971; Gwinner, 1975) and zebra finches (Tueniopygia guttutu custunotis) (Prove, 1978). All of these bird species are however monogamous. The same situation 38 0018-506w91 $1.50

Copyright Q 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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also holds for the Japanese quail (Coturnix cotumix juponicu) (Delville, Sulon, Hendrick, and Balthazart, 1984). It was therefore of interest to repeat some of these experiments on a free-living polygamous bird species, the pied flycatcher (Ficeduh hypoleucu). This species also has an extremely early testicular regression compared to the other bird species studied. On average, the testes of the pied flycatcher are regressed by the time the eggs hatch (Silver-in, 1975). Furthermore, earlier studies have shown that the reproductive behavior of the female pied flycatcher is not prolonged by experimentally prolonging the sexual activities and spermatogenesis of the male (Silver-in, 1980a). During the breeding period, plasma concentrations of LH and testosterone show pronounced seasonal variations (Silverin and Wingfield, 1982). Male pied flycatchers arrive in the breeding area and occupy territories before the females arrive. When a female settles in a territory, she starts nest-building more or less immediately. Within just a day or two, the male is found to have drastically increased LH and testosterone titers, i.e., during the early part of the nest-building period. Territorial but unmated males do not show such a peak. This indicates that the female exerts an effect on the endocrine system of the male which probably plays a role in the synchronization of the reproductive behavior between the male and the female (Wingfield, 1980). The functional significance of this hormonal peak has been discussed by Silverin (1983, 1988). Its function might be to increase the mate-guarding behavior, in order to avoid cuckoldry, and/or to increase courtship behavior, which in turn leads to an increase in female estradiol levels during the later part of the nestbuilding period. Estradiol has been shown to have a key role in initiating sexual behavior in a wide variety of vertebrates (e.g., Feder and Silver, 1974; Tokarz and Crews, 1980; Moore, 1983). The main question in the current study was to find out whether the period of sexual activities in polygynous male pied flycatchers could be prolonged beyond the normal time of testicular regression by giving the females estradiol implants. MATERIALS AND METHODS The present study was conducted during 2 successive years in a deciduous forest, containing large elements of pines and bogs, about 20 km SE of Gothenburg in southwest Sweden. Two study areas situated about 1 km apart were used. The flycatchers in one area were used as a control population, whereas the other population contained the estradiol-treated females. Each study area contained 100 nest-boxes, situated about 30 m apart. During each of the 2 years, about 9598% of all pied flycatchers were given unique combinations of color rings, so that all individuals included in this study could be recognized during field observations. Ringing occurred a couple of days after the birds had arrived at the breeding areas. The exact breeding stage of each bird was known by repeated

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observations of their nests. During the first of these 2 years, the following variables were studied: behavioral responses to simulated territorial intrusions, plasma levels of certain hormones, and organ weights. The following year breeding data were collected and territorial behavior was studied in detail. In the experimental population, all females were implanted subcutaneously with a 6-mm-long silastic tube (i.d. 0.58 in., o.d. 0.77 in.) containing crystalline 17/Sestradiol. Control females were given empty silastic tubes. All implants were given during the nest-building period. Blood samples collected during the nestling period showed that circulating levels of estradiol were about 0.9 rig/ml in the estradiol-treated females (n = 9) and nondetectable in the control females (N = 9). Behavioral

Observations

At different stages in the breeding cycle, flycatcher pairs were subjected to simulated territorial intrusions by placing a stuffed male pied flycatcher (color morph I-II, Drost, 1936) on the roof of the nest-box and by playing conspecific tape-recorded songs through a speaker situated next to the decoy. The distances to some of the neighboring trees were measured in advance. During the behavioral observations there were, at all times, two observers. One observer concentrated his attention on the movements of the male, and the second observer concentrated on the much more inconspicuous female. Behavioral observations were made from a distance of between 10 and 20 m, depending on the vegetation. After the stuffed bird was placed on the roof of the nest-box, a period of 2 min was allowed to elapse before the song and a lo-min-long period of behavioral observations started. During this period, we recorded time of appearance in the territory, distance to the decoy (to the nearest meter) from each of its positions during the period of observation, each movement (flight or jump to a new position), songs and warnings, hovers, attacks (physical attacks on the decoy either by hitting the decoy while hovering, or when sitting on the back of the decoy tearing feathers out of his plumage), and visits to the entrance or into the nest-box; at the end of each 30-set period the distance between the male and the female was estimated. As not all birds spent the entire lo-min period within the territory, behavioral observations are expressed as behavior/min, i.e., presented data cover only the period during which the birds were sighted. It should be noted however that males were observed for the entire time spent in their territories. Unfortunately, due to inconspicuous behavior and dense vegetation, females might have spent some time in the territory before being noticed. The following year no simulated territorial intrusions were performed. After ringing, the birds were left undisturbed. Their territorial behavior was followed daily until the day the clutch hatched. After this date the nest was inspected only every third day. A male was considered monog-

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amous if he, during the entire breeding season, was observed daily in the home-territory and not seen sitting singing at an empty nest-box somewhere else in the study area. A male was considered polyterritorial if he was seen singing, for a day or more, around an empty nest-box (secondary territory) at a time when he already had been paired with a female in another territory (home territory) more than 50 m away, and if he, during 2 successive days, was not sighted in the home-territory. Breeding data presented in this paper were collected during this year. Hormone Assays and Organ Weights

Blood samples were collected from male pied flycatchers at a time when the nestlings had reached an age of 5-6 days. Within l-2 min after capture, a 200-~1 blood sample was collected by a heparinized syringe from the jugular vein. Blood samples were kept on ice until they were centrifuged later the same day. Steroid hormones were later measured by a singleantibody assay after chromatographic separation and purification on microcolumns of celite:glycol, where steroids were eluted in order of polarity. Each sample was assayed in duplicate, and all samples were processed within a single assay to avoid interassay variation. A standard plasma pool from mallards was also included, in duplicate, in the assay. Intraassay variations were testosterone 6%, dihydrotestosterone 12%, LH 11%) and corticosterone only 4%. The method has been described in more detail by Wingfield and Farner (1975), Rdhss and Silverin (1983), and Silverin, Viebke, and Westin (1984). Luteinizing hormone (LH) was measured with the double-antibody radioimmunoassay technique developed by Follett, Scanes, and Cunningham (1972) and Follett, Farner, and Mattocks (1975). In connection with the blood sampling, the body mass of all males and females was recorded to the nearest 0.1 g, using a Mettler spring balance. The amount of stored subcutaneous fat was assessedin the area around and between the wishbone (furculum) using an arbitrary scale from 0 to 5, where 0 = no fat visible, and 5 = a furculum area bulging with fat (for details see Silverin, Viebke, Westin, and Scanes, 1989). Ten males and 10 females from each of the two groups were killed by decapitation, and bodies were fixed in Bouin’s solution. After 24 hr, this fluid was replaced by 70% ethanol. Later organs were weighed on a Mettler precision balance after having dried for 30 set on a paper towel. Testes were sectioned at 7 pm for histological studies. Statistical Analysis

Comparisons between birds from the estradiol group and the control group were made by using a two-tailed Mann-Whitney U test. For the study of seasonal changes in behavioral responses within one group of birds the Wilcoxon matched-pairs signed ranks test was used. Statistical

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TABLE 1 Breeding Data from a Population of Pied Flycatchers, Where Females (n = 23) Received an Empty Silastic Tube during the Early Part of the Nest-Building Period, “Control Population,” and a Population Where the Females (n = 25) Received an Estradiol Implant during the Early Part of the Nest-Building Period, “Experimental Population”

Number of eggs laid Number of eggs hatched Number of fledglings

Experimental population

Control population

6.5 f 0.3 6.3 k 0.4 5.8 f 0.5

6.0 k 0.4 5.9 -c 0.3 5.3 2 0.5

comparisons between proportions of birds performing a certain behavior employed a x2 test. RESULTS Breeding Data The number of eggs laid, number of eggs hatched, and number of fledglings produced in the experimental and control populations are shown in Table 1. In no case was there any difference in these variables between the population containing the estradiol-treated females and the control population. Morphological and Hormonal Data Tables 2 and 3 summarize morphological and hormonal data from the experiment. Data were collected when the nestlings had reached an age of 5 or 6 days. The estradiol-treated females had significantly higher body weights and fat indexes (P < 0.001 in both cases) than control females. No differences in body weight, fat index, or weight of testes were found between males paired with an estradiol-treated female and those paired with a female implanted with an empty silastic tube. Testes from all males were very small. One testis from each male was sectioned at 7 pm, and histological studies showed that all testes had regressed completely. The seminiferous tubules contained only one to two layers of spermatogonia. No mature Leydig cells could be observed in any testis. The ovaries of females were also fully regressed in both groups. However, estradioltreated females had very big oviducts (P < 0.001). Hormonal levels were examined only in the males (Table 3). Males paired with estradiol-treated females had significantly lower plasma levels of corticosterone (P < 0.05) than males paired with control females. No differences between the two groups of males were found in circulating levels of testosterone, DHT, or LH.

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TREATMENT

TABLE 2 Some Morphological Data from Male and Female Pied Flycatchers in a Control and an Experimental (Females Are Estradiol Treated) Population Experimental population Male Body weight (g) Fat index Gonad weight (mg) (n = 10) Oviduct weight (mg) (n = 10)

12.7 k (n = 0.9 f (n = 2.5 f

0.2 20) 0.2 20) 0.8”

Control population

Female

Male

Female

14.1 _’ 0.3 (n = 20) 2.0 + 0.1 (n = 20) 3.0 k 0.5

12.4 + 0.2 (n = 26) 1.0 + 0.2 (n = 26) 3.4 f 0.7

12.5 f 0.2 (n = 26) 0.6 + 0.2 (n = 26) 3.5 2 0.3

170.3 I?r 11.8

9.3 If: 0.9

Note. Data (means &SE) were collected when the nestlings were between 5 and 6 days old. ’ Weight of the left testis.

Behavioral Responsesto a Simulated Territorial Intrusion No seasonal changes in female behavior were observed. Differences were, however, found between estradiol-treated and control females (Fig. 1). Most control females approached the decoy and were hovering in front of the stuffed male sometime during the lo-min observation period. This behavior was never observed among the estradiol-treated females (P < 0.001). In no case was a female seen attacking the decoy. At all times during the breeding cycle, control females changed positions (by jumping to another part of the twig or by flying to another tree or twig) more frequently than did the estradiol-treated females (P < 0.03 TABLE 3 Plasma Levels of Some Hormones in Free-Living Male Pied Flycatchers from a Control Population and a Population Where the Females Had Been Given Estradiol Implants during the Early Part of the Nest-Building Period Experimental population (n = 20) Testosterone (pg/ml) Dihydrotestosterone (pg/ml) LH Wml) Corticosterone (rig/ml)

272 134 0.20 8.8

k + + f

37 7 0.03 1.1

Control population (n = 26) 276 156 0.22 11.5

+ * 2 +

76 49 0.03 0.8

Note. Samples were collected when the nestlings had reached an age of 5 or 6 days. Values are given as means *SE.

BENGT SILVERIN

44

LZ OZ

0.2

q

= CONTROL FEMALES

m

= ESTRAOIOL

TREATED

FEMALES

I I

I I I I I I -r I I I I

t

I

I I I lI I

I I

FIG. 1. Seasonal changes in some behavioral responses of free-living female pied flycatchers to a simulated territorial intrusion by a male and the effects of experimentally increased plasma levels of estradiol in the females. Values are given as means -C SE. Control females, n = 20; experimental females, n = 26.

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TREATMENT

TABLE 4 Numbers and Proportions of Territory Holders and Females Showing Up in the Home Territory during a IO-Mm Simulated Territorial Intrusion by a “Foreign” Male Experimental population

Control population

Breeding stage

Male

Female

Male

Female

Pre-nest-building Nest-building Egg-laying Incubation Nestling

16 (80%) 10 (50%) 20 (100%)

9 (45%) 19 (95%) 20 (100%)

26 (100%) 13 (50%) 11 (42%) 21 (81%) 26 (100%)

18 (69%) 18 (69%) 25 (96%) 26 (100%)

Note. Number of observation periods (= number of nests): experimental population n = 20; control population n = 26. Nests where only the female was feeding the nestlings are not included in this table.

at all times). Control females also had a tendency to stay closer to the intruder. This difference was however significant only during the incubation period (P < 0.03). Otherwise, no differences were found between the two categories of females. The proportion of females showing up in the territory during the observation period is shown in Table 4. No differences were found between experimental and control females. Nor were there any differences in the latency of the response (see Fig. 1) to the intruder. On average, females showed up within l-2.5 min after the onset of the observation period. Males showed both seasonal changes and differences between groups in their behavioral responses to a simulated territorial intrusion (Fig. 2). Control males showed an increase in the latency to respond between the pre-nest-building period and the nest-building period (P < 0.05) and between the latter period and the egg-laying period (P < 0.001). During the incubation period, they appeared much quicker than during the egglaying period (P < 0.05). During the egg-laying (P < 0.001) and the incubation periods (P < 0.05), experimental males responded to the intruders significantly faster than did control males. When comparing this variable between males and females, no differences were found between males and females in the experimental population. Both sexes showed up in the territory at the same time. However, in the control population, females were sighted significantly earlier than males during the egg-laying and the incubation periods (P < 0.05 in both cases). No differences were observed during other periods. Although experimental males returned to the home-territory faster than did control males during egg-laying and incubation periods, they kept a significantly larger distance from the intruder than did control males (P < 0.05 during both periods). This was correlated with the observation

= CONTROL MALES MALES PAIRED WITH ESTRADIOL TREATED FEMALES 5

FIG. 2. Seasonal changes in some behavioral responses of free-living male pied flycatchers to a simulated territorial intrusion by another male and the effects of the presence of an estradiol-treated female. Values are given as means f SE, or proportions (%). Control males, n = 26; Experimental males, n = 20. The histograms showing number of attacks/min only include data from attacking males.

0

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that, during these two periods, the distance between male and female was significantly shorter in experimental pairs than in control pairs (P < 0.01 during both periods). Even during the nestling period, there was a significant difference in this variable between the two groups (P < 0.05). The shortest distance between the intruder and the territory-holder was seen in control males during the pre-nest-building period (P < 0.02, compared to all other groups). The proportion of control males physically attacking the intruder was high during the pre-nest-building period but decreased significantly during the nest-building period (P < 0.001). The frequency of attacks (number of attacks/min) also decreased significantly between these two periods (P < 0.01). The proportion of control males attacking the decoy did not change after the nest-building period. However, there was a change in the frequency of attacks by those males that did attack the decoy. During the egg-laying period, the frequency of attacks was just as low as during the nest-building period, but it increased significantly during the incubation period (P < 0.05) and stayed high in males feeding their nestlings. Significantly more males paired with an estradiol-treated female than control males attacked the decoy during both the egg-laying period (P < 0.01) and the incubation period (P < 0.05). No difference was found during the nestling period. However, among males which attacked the decoy, there was no difference between experimental and control males in the frequency of attacks. Consequently, during the egg-laying period, attacking males from the experimental population showed a low-attack frequency. During the incubation period, the number of attacks/min increased significantly in attacking males (P < O.Ol), and it stayed high during the nestling period. Both control and experimental males often hovered in front of the decoy without attacking it. However, there was no seasonal change in this behavior, nor were there any differences in the frequency of this behavior between the two groups. The number of hovers/min averaged 0.1 to 0.25. Prominent features in the breeding behavior of male pied flycatchers are nest-hole demonstrations and visits inside the nest-box. A nest-hole demonstration is not always followed by a visit inside the nest-box. Only data on visits inside the nest-box are shown in Fig. 2. This behavior occurred frequently in control males during the pre-nest-building and nestbuilding periods. However, once the egg-laying period was reached, the frequency of this behavior decreased significantly (P < O.Ol), and it remained low for the rest of the breeding period. During the egg-laying period, males paired with estradiol-treated females showed this behavior more frequently than did control males (P < 0.01). The frequency of this behavior decreased significantly in the experimental males during incu-

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TABLE 5 Numbers and Proportions (%) of Polyterritorial Males in a Control Population (Number of Breeding Males = 23) and an Experimental Population (Number of Breeding Males = 25) Where the Females Were Given Estradiol Implants during the Early Part of the NestBuilding Period Breeding stage Egg-laying Midincubation Early-nestling

Experimental population

Control population

13 (52%) 9 (36%) 0 (0%)

19 (83%) 5 (23%) 0 (0%)

bation (P < O.Ol), and there was no longer any difference between the two groups of males. Effects on Territorial

Behavior

The territorial behavior of males paired with estradiol-treated females and control males is summarized in Tables 4, 5, and 6. Data in Table 4 are from the first year of this study, and Tables 5 and 6 include data collected during the second year, when no simulated territorial intrusions were performed. As seen in Table 4, both groups of males showed seasonal changes in the number of territorial males showing up in their hometerritory during a simulated territorial intrusion. All control males were present during the pre-nest-building period, but the number showing up was drastically reduced during the nest-building and egg-laying periods (P < 0.001). During egg-laying, significantly more males from the experimental population showed up in the home-territory (P < 0.02). At this stage in the breeding cycle, there was no difference between estradioltreated females and control females. During the incubation period, experimental and control males showed quite the opposite pattern. The number of experimental males showing up decreased significantly (P < 0.05), whereas the number of control males showing up increased significantly (P < 0.01). The number of estradiol-treated females and control females showing up increased significantly between these two periods (P < 0.02 for both groups). During the nestling period all males and females were present at the nest. As seen in Table 5, significantly fewer experimental males established secondary territories during the egg-laying period (P < 0.05). Significantly more control males (P < 0.02) returned to their home-territories at the time when the female in the home-territory was halfway through the incubation period. Experimental males establishing secondary territories left their home-territories on Day 3 k 0.8 of the egg-laying period, and control males on Day 3 k 0.7 of the egg-laying period. Polyterritorial control males returned to their home-territories on Day 8 + 1.4 of the

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TABLE 6 Numbers and Proportions of Monoterritorial Males and Nests Where Only the Females Feed the Nestling (Equals the Number of Males Who Succeeded in Getting a Second Female to Breed in a Secondary Territory)

Monoterritorial males” Females without a feeding maleb

Experimental population

Control population

12/25 (48%) l/26 (4%)

4/23 (17%) 8/31 (26%)

n n/N, n = number of monoterritorial males; N = number of nests where both parents fed their nestlings. b n/N, n = number of single females; N = total number of nests in the population.

incubation period, and experimental males on Day 10 ? 1.7 of the incubation period. Males not having established a secondary territory by the end of the egg-laying period remained monoterritorial throughout the breeding season (Table 6). Thus, the experimental population contained a significantly higher proportion of monoterritorial males than did the control population (P < 0.05). As seen in Table 6, the success of the polyterritorial males differed significantly between the two populations (P < 0.05). In only one case did a male from the experimental population succeed in getting a second female to breed. DISCUSSION Estradiol treatment of female pied flycatchers caused marked changes in the behavior of the male. However, the gonadal cycle of males paired with estradiol-treated females was not prolonged. In the present experiment, estradiol treatment affected the types of behavior that could be observed far less in the female than the behavior of her mate. The reason why there is no behavioral data on the sexual activity of the female is that she lives a quiet, inconspicuous life in the dense foliage of the trees, which makes it more or less impossible, under the prevailing observation circumstances, to observe her behavior toward an intruder trying to copulate with her. Besides, male mate-guarding should prevent her from extra-pair copulations. The treatment did not affect the reproductive success of the pair. For monogamous bird species environmental and behavioral factors play a significant role in the timing of the termination of the breeding season, indicated in the male by testicular regression. The importance of these factors are, for example, reflected in the fact that the onset of gonadal regression may vary by as much as 6 weeks in free-living white-crowned sparrows (Wingfield and Farner, 1979). Behavioral cues from the female may function to synchronize the breeding status of the male with that of

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the female. If females are treated with estradiol their sexual receptivity is increased (e.g., Balthazart and Stevens, 1976; Tokartz and Crews, 1980; Moore, 1983). Prolonging the period of sexual receptivity in female song sparrows, by giving estradiol implants at an early stage of the breeding cycle, results in a prolonged period of intense reproductive and territorial behavior in their mates, as well as prolonged high plasma testosterone levels. The period of high sexual activity can be prolonged by as much as 3 months (Runfeldt and Wingfield, 1985). Similar studies on free-living white-crowned sparrows showed that the normal decrease in male testosterone levels, which occurs during the incubation period, was prevented by giving females estradiol implants (Moore, 1982). Other studies by Moore (1983) showed that male white-crowned sparrows exposed to long days and caged together with estradiol-treated females maintained enlarged testes for a longer period of time than did males caged with control females. However, male responses to female behavioral stimuli are of course dependent upon the ecology of the particular species. The willow tit is another monogamous and, contrary to the white-crowned sparrow, sedentary bird that defends a winter-territory. Normally willow tits pair during the autumn, and they will breed somewhere within the boundaries of their winter-territories. If a photosensitive male willow tit is exposed to long days in the presence of a female (although not treated with estradiol), testes regress much earlier, and plasma levels of LH decrease much earlier, than in males kept alone (Westin, 1989). Thus, it is quite clear that behavioral cues from the female may have profound effects on the duration of reproductive activities of the male, at least in monogamous bird species. The main purpose of the present study was to determine whether the gonadal cycle of the male pied flycatcher could be prolonged in a way similar to that observed in monogamous song and white-crowned sparrows. The results clearly showed that the period of intense sexual behavior was not prolonged, nor was the period of sperm production or high plasma levels of testosterone or LH prolonged beyond the normal time of testicular regression by giving the females a long-acting estradiol implant. It can be argued that another hormonal result could have been determined if more than one sampling occasion had been used. As judged by the appearance of the Leydig cells this is however not likely. Furthermore, the sample size is quite large and sampling time was spread out over almost two weeks (nestlings were in all cases 5 or 6 days) and in no case was one single individual with a high testosterone titer found. These results are consistent with the results from an earlier study that showed that the period of sexual activities in female pied flycatchers could not be prolonged by giving the male a long-acting testosterone implant (Silverin, 1980a,b; Silverin, 1989). This testosterone treatment prolonged territorial and

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courtship behavior and spermatogenesis for months, although males had no successin getting a second female to breed after the normal hatching time of the population. Male behavior, known to promote sexual activities in the female prior to hatching, did not elicit any responses from unmated females during the nestling period or in females whose broods had been robbed immediately after hatching. The lack of an effect of the male on females agrees with the findings on song sparrows (Runfeldt and Wingfield, 1985). In the present study, the same pattern was seen when the female was treated instead of the male. Thus, the length of the sexually active period seems to be fixed in the pied flycatcher. The inability to prolong the sexually active period appears to be related to a number of ecological factors. Flycatchers need time to prepare for migration (Silverin, 1980a,b). The pied flycatcher starts breeding very late (mid-May) in the season. In no other species studied is the seasonal decline in clutch size as rapid as in the pied flycatcher (von Haartman, 1967), and in very few other species studied, e.g., the Emperor penguin (Groscolas, Jallageas, Goldsmith, and Assenmacher, 1986), does testicular regression occur as early in the breeding cycle (around the day the eggs hatch) as it does in the pied flycatcher (Silverin, 1975). Pied flycatchers often start moulting while breeding, occasionally as early as during the egg-laying period. By the end of August, flycatchers start autumn migration. Although being a long distance migrant (central Africa), they may still be moulting when leaving Scandinavia, and they have not formed any fat depots (Creutz, 1955; HyytiH and Vikberg, 1973; Silverin, 1981; Ojanen and Orell, 1982). Prolonged breeding would most likely make the birds even less prepared for autumn migration by delaying the moult and/or onset of migration. Thus, natural selection must have favored a short period of sexual activity for both sexes. Gwinner (1989) demonstrated that pied flycatchers undergo an annual gonadal cycle under the photo regime prevailing in the wintering area (10”N). It could even be that the timing of gonadal regression in pied flycatchers is in fact regulated by the photoperiod in the wintering area. The dual role of increasing day lengths switching “on” and “off” components of the reproductive system has been reviewed by Nicholls, Goldsmith, and Dawson (1988). Quite obviously there is an adaptive value in the polyterritorial behavior of male pied flycatchers as successful males produce more offspring than monogamous male pied flycatchers do. Usually, the evolution of polyterritoriality is only explained from an ecological point of view (e.g., Slagsvold and Lifjeld, 1988). However, it is most likely that, at least for pied flycatchers, the extremely early testicular regression is an important factor that also should be taken into consideration when discussing the evolution of the polyterritorial behavior. The reverse situation, i.e., that

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the short period of sexual activity evolved as a consequence of the polygamous behavior, seems less likely because of the short period of time available for the adults to combine both breeding and moulting. It is, however, difficult to explain why females mate with already mated males. Several hypotheses have been put forward to explain this circumstance. The female mates with an already mated male because he deceives the female by concealing his marital status (e.g., Alatalo and Lundberg, 1984; Temrin and Arak, 1989), and/or because she is subjected to energy constraints when searching for an unmated male (Alatalo, Carlson, and Lundberg, 1987a; Slagsvold, Lifjeld, Stenmark, and Breiehagen, 1988). Furthermore, nest sites are normally limited in a forest, resulting in heavy competition for the ones available. Thus, she cannot spend too much time searching for a good nest site. If she does, she runs a great risk of not mating at all (e.g., Harvey, Stenning, and Campbell, 1985; Nyholm, 1986). Because a female is unable to predict whether the male will assist her or not in feeding the nestlings, it has been argued that if there is a variation in territorial quality, female pied flycatchers choose breeding sites based on this variation, not because of male characteristics (Alatalo, Lundberg, and Glynn, 1986) whereas in a more homogenous habitat she selects her breeding site according to male characteristics (Lifjeld and Slagsvold, 1988). Although the sexually active period of males was not prolonged as a result of estradiol treatment of the females, male behavior during the earlier stages of the breeding cycle was very much affected. For example, males paired with estradiol-treated females responded to the simulated intrusion much earlier than control males. Experimental males kept further away from the decoy, but much closer to their mates, than did the control males. Although there were no differences in “restlessness” between the two groups of males, or in the frequency of attacks between experimental and control males attacking the decoy, significantly more of the experimental males did attack. Thus, the aggressive and mate-guarding behavior of the male was increased as a result of the experimental manipulation. Because of the high reproductive benefits involved in extra-pair copulations for males, they will try to copulate with more than one female, especially females in neighboring territories. On the other hand, a male reduces his reproductive success if he allows his mate to copulate with other males. Consequently, he should try to keep intruders away from the territory, and guard the female, wherever she goes, while she is sexually receptive. For pied flycatchers it is well known that extra-pair copulations occur, although with a low frequency (Bjorklund and Westman, 1983; Alatalo, Gottlander, and Lundberg, 1987b). The extent to which these copulations lead to extra-pair fertilizations is debated (Lifjeld and Slagsvold, 1989). The results of the present study support the idea

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that cuckoldry is a problem for the territory holder. Copulations among pied flycatchers are most frequent during the days before the onset of egg-laying (von Haartman, 1951; Alatalo et al., 1987b), indicating that the female is most receptive during this period. At the time when males establish their secondary territories very few copulation attempts have been observed. This correlates with my observation that the distance between control males and females increased between nest-building and egg-laying, whereas experimental males maintained the same distance from their mates during the egg-laying period as the control males did during nest-building. The fact that the distance between experimental males and females was always significantly shorter than between individuals in control pairs further indicates the importance of mate-guarding behavior. Furthermore, as significantly more of the males mated with estradiol-treated females remained monoterritorial, and as many of the polyterritorial males also showed up in their home-territories during the simulated intrusions, and as experimental males also appeared earlier at the nest than did the control males, it is likely that being cuckolded really is a problem for the territory holder, at least during the period when the female is sexually receptive. Males paired with such females really have to be alert. In earlier papers I have claimed that male pied flycatchers become monoterritorial if the plasma level of testosterone decreased to baseline during the egg-laying period, as a result of lack of supplementary stimuli from unpaired females (Silverin, 1988). In the present experiment, significantly more experimental males than control males remained monoterritorial. Unfortunately no blood samples were collected from these males, so it can only be speculated that these males were monoterritorial because of low testosterone levels at this time of the breeding cycle. However, another viable hypothesis is that if the females showed signs of receptivity, males would be expected to remain monoterritorial and mate guard despite high testosterone levels. Termination of receptivity could be a signal to the male that he can leave without risk to his paternity of the clutch. This hypothesis could be supported by the observed increase in mate guarding by males paired with estradiol-treated females. Whether a male pied flycatcher attacks a territorial intruder or not, or whether he attacks with a high or low frequency, is independent of the plasma testosterone levels. Even male flycatchers from the nestling period with basal levels of testosterone may be very aggressive, while other males with the same concentration of testosterone and in the same breeding stage can be completely uninterested in the decoy during a simulated territorial intrusion (Silverin, unpublished data). Thus, aggressiveness can be controlled by factors other than plasma levels of testosterone. The experimental population contained very few unpaired females, as indicated by the very low success of polyterritorial males in getting a

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second female to breed. As shown in the present study females do not attack male intruders. However, they will attack other females that appear on their territories (Breiehagen and Slagsvold, 1987), and the aggressive behavior of females is especially pronounced during nest-building and egg-laying, i.e., when females normally have increased plasma levels of estradiol (Silverin and Wingfield, 1982). Breiehagen and Slagsvold also showed that primary females chased away females in secondary territories situated more than 100 m away from home-territories. Thus, the low success of polyterritorial males paired with estradiol-treated females to mate with a second female might be a result of aggressive encounters between females. Unpaired females may be chased away from the area. The possibility that a lack of unpaired females in the breeding area was more or less responsible for the high proportion of monogamous males observed in the experimental population cannot be excluded. ACKNOWLEDGMENTS This study was supported by a grant from the Swedish Natural Science Research Council (B-BU 4342-114), and by grants from the following foundations: Anna Ahrenbergs fond for vetenskapliga Bndamil, Stiftelsen Lars Hiertas minne, and the Kungliga och Hvitfeldtska stipendieinrlttningen. The field assistance of Conny Olsson and Fredrik Silverin is gratefully acknowledged.

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