Age of Clutches in Nests and the Within-Nest Spawning-Site ...online.sfsu.edu/vancev/Vredenburg_Lab/Publications...KNAPP ET AL.-CLUTCH DEPOSITION PATTERNS IN DAMSELFISH 79 have important

Age of Clutches in Nests and the Within-Nest Spawning-Site Preferences of ThreeDamselfish Species (Pomacentridae)

Roland A. Knapp; Paul C. Sikkel; Vance T. Vredenburg

Copeia, Vol. 1995, No. 1. (Feb. 15, 1995), pp. 78-88.

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CopPia, 1995(1), pp. 78-88

Age of Clutches in Nests and the Within-Nest Spawning-Site Preferences of Three Damselfish Species (Pomacentridae)

In numerous fish species, males provide exclusive parental care of the off- spring, and females often use male or nest characteristics to discriminate among potential nests. Little attention, however, has focused on the possibility that once females have chosen a nest, they may also make choices regarding where in the nest to lay their eggs. In this paper, we describe within-nest clutch-layingpatterns in three damselfish species, Stegastes partitus, S. leucostictus, and Hypsypops rub-icundus. In all three species, females prefer to deposit clutches contiguous to early-stage clutches and noncontiguous to late-stage clutches, even when early- and late-stage clutches are only a single day apart in age. This pattern holds true both in nests containing multiple clutches and in nests containing single clutches. Despite these within-nest spawning-site preferences of females, we were unable to detect an effect of clutch location on clutch survival in S. partitus. We examined several explanations for the female within-nest spawning site preferences. These were as follows: (1) the observed egg-laying patterns reduce the risk of egg predation through a dilution effect; (2) clutches of different ages have different metabolic requirements, and the observed egg-laying patterns reduce competition (e.g., for oxygen) between clutches; (3) the within-nest preferences are a conse- quence of a between-nest preference for early-stage clutches; and (4)the observed egg-laying patterns are a response to patterns of male parental care (e.g., males may provide more care to younger clutches). We conclude that this "differential care" hypothesis is the most likely explanation for the female within-nest spawn- ing-site preferences.

EXCLUSIVE male care of eggs is the most male or nest features result in increased off- common form of parental care in teleost spring survival (e.g., CBte and Hunte, 1989;

fishes, occurring in approximately 76% of fam- Knapp and Kovach, 199 1 ; Knapp, 1993). ilies that have some form of care (Blumer, 1982; Despite the interest in elucidating male or Gross and Sargent, 1985). In the majority of nest characteristics that females use & discrim-fish species with male parental care, females lay inating among nests, researchers have largely eggs in nests within male territories. Males de- ignored the possibility that females may choose fend the eggs and, in some species, newly where in a nest to lay their eggs once they have hatched offspring, for several days to weeks chosen a nest. For example, parental males of (Breder and Rosen, 1966). These fishes have many fish species often acquire clutches from attracted the attention of numerous researchers several females over a period of several days or interested in female mate choice and sexual se- weeks (Breder and Rosen, 1966). Having cho- lection, in part because of the relative ease with sen a nest in which to sDawn, females mav have which male reproductive success can be quan- the opportunity to deposit their clutches non- tified and because parental males have the po- randomly with respect to the ages of clutches tential to strongly influence egg survival (Wil- already present in a nest. We are aware of only liams, 1966; Trivers, 1972). Studies of female a single paper that addresses such within-nest mate choice in s~ecies with male ~ a r e n t a l care spawning-site preferences. Sikkel (1989) dem- have often demonstrated female preferences for onstrated experimentally that when female gar- male characteristics (e.g., Semler, 197 1; Bisazza ibaldi (Hjpsjpops rubicundus) are given access to and Marconato, 1988; Knapp and Warner, 199 1) both early- and late-stage clutches within a sin- and nest characteristics, including nest size (Bis- gle nest, they prefer to lay their eggs among azza et al., 1989), concealment (Sargent, 1982), early-stage clutches. In addition to the paucity substratum type (Sikkel, 1988), number of eggs of data on within-nest spawning-site prefer- present (e.g., Marconato and Bisazza, 1986; Un- ences in other species, there have been no at- ger and Sargent, 1988; Gronell, 1989), and age tempts to determine why females might show of eggs present (Sikkel, 1989). Several studies such preferences. have also shown that female preferences for Within-nest spawning-site preferences may

O 1995 by the Amerlcan Society of Ichthyolog~sts and Herpetologists

79 KNAPP ET AL.-CLUTCH DEPOSITION PATTERNS IN DAMSELFISH

have important consequences for mating sys- tems as a result of the effect of these preferences on male and female fitness. Male fitness could be affected in at least two ways. First, if females prefer to lay clutches contiguous to particular types of clutches, females may prefer to spawn in nests containing the preferred clutch type. Males defending nests with the preferred clutch type would experience enhanced mating success (Sikkel, 1989). Second, within-nest preferences may affect how many clutches a nest can accom- modate (Sikkel, 1994). Female fitness could also be affected in at least two ways. First, female fitness may increase as a result of within-nest spawning-site preferences if these preferences result in enhanced clutch survival. Second, vari- ance in fitness among females could be high if differences among nests in clutch survivallead to intrasexual competition among females for access to high-quality nests (Rosenqvist, 1990).

In this paper, we present data on within-nest spawninipatterns fbr three damselfish species: the bicolor damselfish (Stegastes partitus), beau- gregory damselfish (Stegastes leucostictus), and garibaldi (Hjpsjpops rubicundus). We extend the previous work on H. rubicundus (Sikkel, 1989) by presenting more detailed information on fe- male spawning patterns in both unmanipulated and manipulated nests. Our results show that, within nests, females of all three s~ecies lav their eggs nonrandomly with respect to the age of other clutches present, preferring to lay eggs among early-stage clutches and away from late- stage clutches. In addition to describing the fe- male egg-laying patterns, we use data on clutch position and hatching success in S, partitus to test the hypothesis t h a t female within-nest spawning-site preferences result in increased iffspring survival.

Study animals.-The bicolor damselfish, S. par- titus, and beaugregory damselfish, S, leucostzctus, are small pomacentrids common on coral reefs throughout the Caribbean (S. partitus: < 8 cm TL; S. leucostictus: < 11 cm TL; Randall, 1968). The breeding biology of both species is de- scribed in detail elsewhere (S. partitus: Myrberg, 1972; Schmale, 198 1; Knapp and Warner, 1991; S. leucostictus: Itzkowitz and Makie, 1986; Rob- ertson et al., 1990; Itzkowitz, 1991). Males and females of both species are permanently terri- torial; and within their territories, males main- tain a smooth-walled coral surface on which fe- males deposit adhesive eggs. Reproduction is lunar cyclic (Robertson et al., 1990). Eggs are laid in a monolayer; and although more than one female may spawn in a nest simultaneously, all eggs laid on a single day are deposited into

a discrete clutch. Because males of both species do not undergo brood cycles (i.e., a distinct mat- ing phase followed by a brooding phase; van Iersal, 1953), successful males often mate daily during a reproductive cycle and nests usually contain multiple clutches on any given day. In S, partitus, newly laid eggs are white, 1-day-old eggs are dull yellow, 2-day-old eggs are pink, and 3-day old eggs are dark purple (Knapp and Warner, 1991). In S. leucostictus, newly laid eggs are bright yellow, 3-to 4-day-old eggs are yel- low-brown, and 6-day-old eggs are dark purple (Itzkowitz and Makie, 1986). Males care for eggs until the eggs hatch as planktonic larvae, 3.5 days after laying in S. partitus (Robertson et al., 1988) and 4.5-5.5 days after laying in S. leucos- tictus (VTV and RAK, pers. obs.).

The garibaldi (H. rubicundus) is a large (< 36 cm TL) damselfish common in rocky subtidal areas of southern and Baja California (Lim- baugh, 1964; Clarke, 1970). The breeding bi- ology of this species is described in detail else- where (Clarke, 1970; Sikkel, 1988, 1989). Both sexes defend permanent territories. During the spring-summer spawning season, males pro-mote the growth of a nest of red algae on an open rock face within their territories (Foster, 1972). Females deposit adhesive eggs in nests; eggs are bright yellow when first deposited and turn gray after approximately five days. Males defend eggs until the eggs hatch after 12-23 days (Sikkel, 1988). Females prefer to spawn in nests that contain early-stage eggs (Sikkel, 1 989). Therefore, although receptive males may take weeks to receive an initial clutch, once males receive eggs, most spawn daily with numerous females during the next 5-10 days. This mating phase is followed by a brooding phase during which males care foi eggs but d i not spawn. A complete brood cycle lasts 20-30 days, and males maicomplete up to four brood cycles in a sea- son.

Patterns of clutch deposztion: Stegastes partitus.- Data on S. partitus were collected by RAK on the Teague Bay fore reef, Saint Croix, United States Virgin Islands (1 7"45'N, 64"35'W) during a total of four reproductive cycles between 1989 and 1990. Prior to data collection, the natural nests of approximately 25 males were replaced with small clay flowerpots (13 cm diameter) as part of a concurrent study of mate choice in this species (Knapp and Kovach, 199 1; Knapp and Warner, 199 1; Knapp, 1993). All males switched their activities to the artificial nests within hours of nest replacement, and within-nest egg-laying

80 COPEIA, 1995, NO. 1

patterns were identical between natural and ar- tificial nests (RAK, pers, obs.). During each re- productive cycle, we checked all nests daily for the presence of new clutches (0 days old); when present, we recorded their location rel- ative to other clutches in the nest. Clutches were identified as 0, 1, 2, or 3 days old using age- specific pigmentation and clutch location. A clutch was termed "contiguous" if it was laid so that it touched another clutch anywhere along its perimeter. Fewer than 1% of all clutches were laid contiguous to more than a single clutch.

All clutches present in nests appeared acces- sible to egg-laying females, since clutches always had at least 50% of their perimeter exposed. In addition, because nests were never space lim- ited, females always had the option of laying contiguously or noncontiguously to any clutch in the nest. However, to ensure that the ob- served spawning-site preferences did not result from differential access to clutches of various ages (e.g., if 1-day-old clutches were more or less accessible than 2- or 3-day-old clutches), we also analyzed female clutch-laying patterns in those nests that contained only single clutches (1,2, or 3 days old). In such nests, females clear- ly had the choice of laying contiguously or non- contiguously to the single clutch present. The results described below are based on the posi- tions of 638 clutches laid by approximately 60 females.

Patterns of clutch deposition: Stegastes leucostic-tus.-Data on S. leucostictus were collected by VTV and RAK on the Teague Bay back reef during two reproductive cycles in 1991. Prior to data collection, we replaced the natural nests of 20 males with artificial nests identical to those used with S. partitus. All males switched their activities to the artificial nests within hours of nest replacement, and within-nest egg-laying patterns were identical between natural and ar- tificial nests (VTV and RAK, pers. obs.). We checked all nests daily for the presence of new clutches; when present, we recorded their lo- cation relative to other clutches in the nest. Clutches were identified as 0, 1, 2, or 2 3 days old using age-specific pigmentation and clutch location. A clutch was termed "contiguous" if it was laid so that it touched another clutch anywhere along its perimeter. Fewer than 10% of all clutches were laid contiguously to more than a single clutch.

~ t e ~ a s t e sleucostictus nests were rarely space limited. However, in 37 of 478 cases (7.7%). , ,- , ,

females spawned in nests in which the youngest preexisting clutch was inaccessible. Although

such cases mav weaken anv within-nest clutch- ,deposition patterns, we included all observed clutch positions in our analyses to avoid any unforeseen biases resulting frdm excluding these cases.

As with S. partitus, we also analyzed female clutch-laying patterns in nests containing only single clutches (1, 2, or r 3 days old) to ensure that the observed spawning-site preferences did not result from differential access to clutches of various ages. The results presented below for S. leucostictus are based on 478 clutches laid by at least 30 females.

Patterns of clutch deposition: Hypsypops rubicun- dm.-Data on H. rubicundus were collected be- tween 1986 and 199 1 by PCS in Big Fisherman Cove, Santa Catalina Island, California (33"28'N, 1 18"2gfW). A total of 79 nests were monitored during this period. Nests were checked daily for the presence of new clutches; when present, their location relative to other clutches in the nest was recorded. As with S. partitus and S. leucostictus, a clutch was termed "contiguous" if it was laid so that it touched another clutch anywhere along its perimeter. Clutches were identified as early stage (0, 1, or 2 days old) or late stage ( r 3 days old) by using age-specific pigmentation (see Sikkel, 1988) and maps of clutch positions from the previous day.

Female H. rubicundus prefer to deposit eggs around early-stage clutches (Sikkel, 1988; see Results). Because of the manner in which eggs are deposited, clutches generally accumulate such that only the youngest clutch is exposed to empty nest space (Sikkel, 1994). As a result, situations in which only late-stage eggs are ex- posed to empty nest space occur only rarely, and situations in which females have access to clutches of different ages are also uncommon. As part of a study on filial cannibalism in H. rubicundus, PCS conducted experiments in which eggs were removed from broods to make nest space available adjacent to early-stage, late-stage, or early- and late-stage clutches (Sikkel, 1994). T h e position of the first clutch laid subsequently in each experimental nest was recorded during daily nest visits. The results presented below are based on the positions of 614 clutches laid in unmanipulated nests and 233 clutches laid in experimental nests. These clutches were laid by at least 90 females.

Survival of clutches i n relation to clutch position.-- Because female S , partitus strongly prefer to lay contiguously to 1-day-old clutches and noncon- tiguously to 2- and 3-day-old clutches (see Re- sults), we tested whether these within-nest

KNAPP ET AL.-CLUTCH DEPOSITION PATTERNS IN DAMSELFISH 81

spawning-site preferences have consequences for clutch survival. T o do this, we took advantage of the fact that females occasionally laid clutch- es in the "wrong" position. In nests in which the youngest clutch was 1-day-old, females oc- casionally laid contiguously to 2- or 3-day-old clutches or noncontiguously to any clutches. Similarly, in nests in which the youngest clutch was 2 or 3 days old, females occasionally laid contiguously to these clutches. Therefore, in nests containing a brood whose youngest clutch was 1 day old, we compared the survival of clutches laid contiguously to a 1-day-old clutch ("correct" position) with the survival of clutches laid contiguously to 2- or 3-day-old clutches and clutches laid noncontiguously to any of the clutches present ("wrong" positions). Given the hypothesis that female within-nest spawning-site preferences result in increased clutch survival, h e predicted that clutches laid contiguously to 1-day-old clutches would have higher survival than' those clutches laid either co~tiguously to 2- or 3-day-old clutches or noncontiguously to any of the bailable clutches. In nests containing a brood whose youngest clutch was 2 or 3 days old, we compa;ed tha survival of clutches laid noncontiguously to 2- and 3-day-old clutches ("correct" posit'ion) with the survival of clutches laid contiguously to any of the clutches present ("wrong" position). Because females prefer to lay noncontiguously to 2- or 3-day-old clutches, we predicted that clutches deposited noncon- tiguously to any clutches would have higher sur- vival than those laid contiguously to 2- or 3-day- old clutches.

We measured the survival of clutches laid in S. partitus nests by tracing the perimeters of any new clutches (0 days old) onto transparent ac- etate sheets during daily nest visits. If a clutch suffered egg losses by the following day, the portion of the clutch remaining was retraced. Clutch areas were quantified using a digitizer (for details, see Knapp and Warner, 1991). We defined clutch survival as that ~ r o ~ o r t i o n of the

I 1

clutch that remained one day after laying. We were unable to measure survival from egg-lay- ing to hatching because the deposition of sub- sequent clutches contiguously to each test clutch would confound any effects of clutch position on egg survival. Because the majority of egg losses involved the loss of the entire clutch (22 complete losses, one loss of > 50%, and seven losses of < 50%), clutches were recorded as lost if > 50% of the original clutch area disappeared by the following day.

Statistical analysis.-We analyzed data on clutch placement as function of clutch age using chi-

square and binomial tests. When nests con-tained multiple clutches, we assumed that fe- males had equal access to all of the clutches and to empty nest space. The null hypothesis we tested was that females laid contiguously to any of the clutches and noncontiguously with equal probability. For example, in a nest containing three clutches, the probability of each laying option (contiguously to any of the clutches and noncontiguously) would be 0.25. Because fe- males sometimes deposited clutches in such a way as to reduce access to older clutches, our assumption of equal clutch access may some-times be violated. Therefore, we also analyzed female clutch laying patterns in nests containing single clutches. The null hypothesis we tested was that females laid contiguously or noncon- tiguously with equal probability. This test re- moves any bias associated with the assumption of equal access to all clutches, but both analyses contain a potential bias associated with the as- sumption that an equal nest area was available for females to lay contiguously or noncontig- uously. In reality, the amount of empty nest space in which females could lay noncontig- uously nearly always exceeded that in which fe- males could lay contiguously. Because egg-lay- ing patterns were very similar between nests with multiple clutches and single clutches (see Results) despite large differences in the amount of empty nest space between the two nest types (less space to lay noncontiguously in nests con- taining multiple clutches), this assumption did not appear to influence our results.

Patterns of clutch deposition: Stegastes partztus.- The age of clutches present in nests had a strong influence on the placement of subsequent clutches by female S. partitus (Fig. 1) . When S. partitus females spawned in nests containing broods in which the youngest clutch was 1 day old(age 1, 1 + 2, 1 + 3 , o r 1 + 2 + 3),signif-icantly more clutches were deposited contigu- ously to 1-day-old clutches than expected if clutches were laid randomly with respect to the ages of other clutches (Fig. 2). Significantly few- er clutches than expected were deposited either contiguously to 2- or 3-day-old clutches or non- contiguously to any of the clutches present. When females spawned in nests containing broods in which the youngest clutch was 2 days old (age 2, 2+3), significantly fewer clutches were deposited contiguously to 2- or 3-day-old clutches than expected, and significantly more clutches than expected were deposited noncon- tiguously to any clutches present (Fig. 2). Sim-

82 COPEIA, 1995, NO. 1

Youngest clutch in nest = 1 day old

Youngest clutch in nest = 2 days old

Fig. 1. Clutch deposition patterns of female Ste- gastespartitus, illustrating the typical position of a new clutch (0 days old) when laid in a nest containing a brood whose youngest clutch is 1 day old and when laid in a nest containing a brood whose youngest clutch is 2 days old. Numbers within each clutch indicate clutch age in days.

ilarly, when females spawned in nests contain- ing only a 3-day-old clutch, significantly fewer clutches were laid contiguously to the 3day-old clutches, and significantly more were laid non- contiguously to the 3-day-old clutches than ex- pected (Fig. 2). Noncontiguous clutches were often deposited on the opposite side of the nest from the 2- and 3-day-old clutches.

The clutch-laying patterns of S. partitus fe- males in nests containing a single clutch mirror those observed in nests containing multiple clutches. Clutch-laying patterns were signifi- cantly heterogeneous with respect to clutch age Or' = 109.8, df = 2, P < 0.0001; Fig. 3). Stegastes partitus females spawning in nests containing a lday-old clutch always laid contiguously to that clutch, whereas females spawning in nests con- taining a 2- or 3-day-old clutch deposited the majority of their clutches noncontiguously to the available clutch. Females were much more likely to lay contiguously when spawning in nests containing a 1-day-old clutch than when spawn- ing in nests containing a 2- or 3-day-old clutch (x* = 109.8, df = 1, P < 0.0001; analysis of subtables, Siegel and Castellan, 1988: 1 18).

I - wnagu& to age 3 1 ' cmtzguour to age z 1 cmt~g~ous to age 1 I

Age= 1 Age=2 Age=3 Age of youngest clutch in nest (days)

Fig. 2. Stacked-bar graph illustrating the actual and expected percentage of clutches female Stegastes partitus laid contiguously to 1-, 2-, or 3day-old clutch- es or laid noncontinuouslv to anv clutches as a func- tion of the age ofyhe y&nges; clutch in the nest. Nests whose youngest clutch was 1 day old contained clutches of age 1, 1 + 2, 1 + 3, or 1 + 2 + 3. Nests whose youngest clutch was 2 days old contained clutches of age 2 or 2 + 3. Nests whose youngest clutch was 3 days old contained only a 3-day-old clutch. Total number of clutches laid in nests of each age category are given at the top of the "actual" bar. ~stei isks above the "actual" and "expected bars in- dicate that actual and expected laying patterns are significantly different (***: P < 0.0001, *: P < 0.025).

However, the strength of the female preference to lay noncontiguously to older clutches was not significantly different when spawning in a nest containing a 2day-old clutch vs a 3-day-old clutch (x2 = 0, df = 1, P > 0.5).

Despite the strong preference by S. partitus females to lay contiguously to 1-day-old clutch- es, females do not appear to use the presence of lday-old clutches in nests to discriminate among potential nests. Females were just as like- ly to spawn in nests containing a brood whose youngest clutch was 1 day old as they were to spawn in nests containing broods whose youn- gest clutch was 2 or 3 days old (x* = 5.2, df = 2, P > 0.05).

Patterns of clutch deposition: Stegastes leucostic- tus.-Within-nest spawning-site preferences of S. leucostictus females were very similar to those shown by S. partitus (Fig. 4). When nests con- tained a 1-day-old clutch (age 1, 1 + 2, 1 + r 3, 1 + 2 + r 3), females deposited more clutch-

- -

--

KNAPP E T AL.-CLUTCH DEPOSITION PATTERNS IN DAMSELFISH 83

-

17contiguous

" . I 65 non contiguous 1 - - I , ;non contiguous

-

---

Age of clutch in nest (days)

Fig. 3. Within-nest clutch-laying patterns of fe- male Stegastespartitus in nests containing single clutch- es of age 1, 2, or 3 days old. Sample sizes are given above each bar. Asterisks adjacent to clusters indicate that the difference between the number of clutches laid contiguously vs noncontiguously are highly sig- nificant (Binomial Test, P < 0.0005).

non-contiguous, con t i g~au~to age 3 contiguous to age 2 1

I-i I Lontiguous to age --

1 I

Age= 1 Age=2 Age23

Age of youngest clutch in nest (days)

Fig. 4. Stacked-bar graph illustrating the actual and expected percentage of clutches female Stegastes l~uco~ t i c tu slaid contiguously to 1-, 2-, or 2 3-day-old clutches or laid noncontiguously to any clutches as a function of the age of the youngest clutch in the nest. Nests whose youngest clutch was 1 day old contained clutches of age 1, 1 + 2, 1 + r 3, or 1 + 2 + r 3. Nests whose youngest clutch was 2 days old contained clutches of age 2 or 2 + r 3. Nests whose youngest clutch was r 3 days old contained one or more clutch- es r 3 days old. Total number of clutches laid in nests of each age category are given at the top of the "ac- tual" bar. Asterisks above the "actual" and "expect- ed" bars indicate that actual and expected laying pat- terns are significantly different (***: P < 0.0001).

Age of clutch in nest (days)

Fig. 5. Within-nest clutch-laying patterns of fe- male Strgastes leucostzctus in nests containing single clutches of age 1, 2, or r 3 days old. Sample sizes are given above each bar. Asterisks adjacent to clusters indicate that the difference between the number of clutches laid contiguously vs noncontiguously are highly significant (Binomial Test, P < 0.0005). T h e lack of asterisks indicates that the difference is not significant (Binomial Test, P > 0.3).

es contiguously to l-day-old clutches, fewer con- tiguously to 2- and ? 3-day-old clutches, and fewer noncontiguously to any of the clutches present than expected under random clutch placement. When nests contained a brood whose youngest clutch was 2 days old (age 2, 2 + I 3) or 2 3 days old (age r 3), females preferred to deposit clutches n ~ n ~ o n t i ~ u o u s l y td the avail- able clutches.

The clutch-laying patterns of S, leucostictus fe-males in nests containing a single clutch were very similar to those observed in nests contain- ing multiple clutches (Fig. 5). Clutch-laying pat- terns were again significantly heterogeneous with respect to clutch age (xZ= 51.6, df = 2, P < 0.000 1).Stegastes leucostictus females Spawning in nests containing a l-day-old clutch nearly al- ways laid contiguously to that clutch, whereas females spawning in nests containing a 2- or 2

3-day-old clutch deposited the majority of their clutdhes nonconti~uously to the prkexisting clutch. Females were much more likely to lay contiguously when spawning in nests containing a l-day-old clutch than when spawning in nests containing a 2- or r 3-day-old clutch (x2= 47.7, df = 1, P < 0.0001). In contrast to the pattern shown by S, partitus, however, spawning-site preferences of S, leucostictus females spawning in nests containing a 2-day-old clutch vs a I 3-day-old clutch were significantly different (x2 = 3.9, df = 1, P < 0.05). Stegastes leucostictus females spawning in nests containing a 2-day- old clutch did not show a statistically significant preference for laying contiguously vs >oncon-

84 COPEIA, 1995, NO. 1

NBSt boundary

Fig. 6. Clutch deposition patterns in Hypqpops rubicundus. Numbers within each clutch indicate clutch age in days. (A) Typical position of a new clutch (0 days old) deposited in a nest in which early-stage clutches (5 2 days old) are exposed to available nest space. The new clutch is laid contiguously to the ex- isting clutches in the nest. (B) Typical position of a new clutch (0 days old) deposited in a nest containing only late-stage clutches (r 3 days old). The new clutch is laid noncontiguously to the clutches present in the nest.

tiguously but strongly preferred to lay noncon- tiguously when spawning in nests containing a clutch r 3 days old (Fig. 5).

In contrast to the between-nest spawning pat- tern shown by S. partitus, S. leucostictus females were more likely to spawn in nests containing a brood whose youngest clutch was 1 day old than in nests containing broods whose youngest clutch was 2 or r 3 days old kP = 22.9, df = 2, P > 0.01). Although there are several con-

ceivable causes of this pattern, one possibility is that females use clutch age to discriminate among potential nests, laying preferentially in those nests containing 1-day-old clutches.

Patterns of clutch deposition: Hypsypops rubicun- dm.-The age of clutches in nests also strongly influenced the position in which female H. rub- icundus deposited subsequent clutches (Fig. 6). Female H. rubicundus laying in unmanipulated nests in which onlv earlv-stane clutches were

8 0

exposed to available nest space always deposited clutches contiguously to early-stage clutches in- stead of depositing clutches noncontiguously (Table 1). When only late-stage clutches were present in unmanipulated nests, females always laid noncontiguously to the available clutches (Table l), often depositing clutches at the o p posite end of the nest. When unmanipulated nests had available nest space adjacent to both early- and late-stage clutches, females again al- ways laid contiguously to the early-stage clutch- es instead of contiguously to late-stage clutches or noncontigu~usi~ to .any of th; available clutches (Table 1).

Egg deposition patterns observed in manip ulated nests were very similar to those in un- manipulated nests (Table 2). When females spawned in nests in which only early-stage clutches were exposed to available nest space, they always deposited clutches contiguously to early-stage clutches. When females spawned in nests where only late-stage clutches were ex- posed, females always laid noncontiguously to the available clutches. When spawning in nests in which both early- and late-stage clutches were exposed to available nest space, females always deposited clutches contiguously to early-stage clutches.

Suruival of clutches in relation to clutch position.- We had survival data for a total of 441 S. partitus clutches that were laid into nests containing broods whose youngest clutch was 1 day old. Of

TABLE 1. WITHIN-NEST CLUTCH-LAYING PATTERNS OF FEMALE Hypsypops rubicundus IN UNMANIPULATED NESTS. "Early-stage" clutches are 5 2 days old, and "late-stage" clutches are 2 3 days old. N.A. = not

applicable.

Female clutch-laying pattern

Contiguous to Contiguous to Not Brood layout earlyatage clutch lateatage clutch contiguous P

Only early-stage clutch exposed 548 N.A. 0 <0.0001* Only late-stage clutches present N.A. 0 12 <0.0005* Early and late-stage clutches exposed 54 0 0 <0.0001t

Binomial test. t Chi-square test.

KNAPP ET AL.-CLUTCH DEPOSITION PATTERNS IN DAMSELFISH 8 5

TABLE2. CLUTCH-LAYING OF FEMALE EGGSWITHIN-NEST PATTERNS Hypsypops rubicundus IN NESTSIN WHICH WERE EXPERIMENTALLY REMOVEDFROM BROODS CLUTCHES NEST SPACE. "Early-TO EXPOSE TO AVAILABLE

stage" clutches are 5 2 days old, and "late-stage" clutches are 2 3 days old. N.A. = not applicable.

Female clutch-laying pattern

Contiguous to Contiguous to Not Brood layout early-stage clutch late-stage clutch contiguous P

Only early-stage clutch exposed 159 N.A. 0 <0.0001* Only late-stage clutch exposed N.A. 0 34 <0.0001* Early and late-stage clutches exposed 40 0 0 <0.0001t

* Binomial test. t Chi-square test.

these, 344 were laid contiguously to a 1 -day-old clutch, and 94.8% survived; 12 were laid con- tiguously to a 2- or 3-day-old clutch, and 9 1.7% survived; and 85 were laid noncontiguously to any clutches, and 95.3% survived. These dif- ferences in survival between clutch position cat- egories were not statistically significant (x2 =

0.69, df = 2, P > 0.5). One-hundred fourty-one clutches were laid into nests containing broods whose youngest clutch was 2 or 3 days old. Of these, 29 were laid contiguously to a 2- or 3-day- old clutch, and 96.6% survived; and 112 were laid noncontiguously to any clutches, and 92.8% survived. Again, these differences in survival were not statistically significant (x2 = 0.52, df = 1, P > 0.3). In addition, the survival of all clutches laid contiguously was not different from the survival of all clutches laid noncontiguously (94.8% and 93.9%, respectively; x2 = 0.21, df = 1, P > 0.5).

The within-nest spawning sites chosen by fe- male S. part i tus , S, leucostictus, and H. rub icundus are strongly influenced by the ages of clutches already present in nests. when early-stage clutches were present in nests (0 days old in S. partitus and S. leucostictus, and 0-2 days old in H. rub icundus) , females preferentially deposited clutches contiguously td them. when oniy later- stage clutches were present in nests, females preferentially deposi'ted clutches noncontig-uously to any clutches present, often laying clutches on the opposite-side of the nest. i'he differential response by S . partitus and S . leucos- tictus females td clutches differing in age by only a single day is particularly remarkable because females of these species gknerally begin spawn- ing before dawn (Robertson et al., 1990; Knapp and Warner, 1991) when light levels are still very low (RAK and VTV, pers. obs.).

Although we have attributed the within-nest spawning patterns observed in all three species to female preferences, clutch deposition pat-

terns may instead be a consequence of spawn- ing-site preferences of males. For example, hav- ing new clutches contiguous to other early- stage clutches may reduce the amount of time required for males to care for them. If this was true, we would expect to see males "directing" females to particular within-nest spawning-sites. This does not appear to be the case in any of the three damselfish used in this study, since females entered nests and began to spawn with- out any obvious interference from resident males. In S. part i tus , females sometimes began spawning while males were outside the nest at- tempting to attract additional females. Males may be able to "direct" females to particular nest locations in a more subtle fashion by se- lectively preparing the nest surface for eggs. This also did not appear to be the case in any of the three study species, since we never ob- served any differences in nest surface charac- teristics in the available nest space. Therefore, clutch deposition patterns are most likely the consequence of preferences by females and not males.

The major difference in within-nest spawn- ing-site preferences between the three species is the clutch age at which females switch from laying contiguously to laying noncontiguously. In S . part i tus , females strongly preferred laying contiguously to 1-day-old clutches and ap-peared to avoid both 2- and 3-day-old clutches. Stegastes leucostictus females also strongly pre- ferred laying contiguously to 1-day-old clutches and appeared to avoid clutches r 3 days old. However, S , leucostzctus females did not show any preference for laying contiguously or noncon- tiguously to 2-day-old clutches. In H. rubicun-d u s , females showed a strong preference to lay contiguously to 0-, 1-, and 2-day-old clutches and appeared to avoid clutches 2 3 days old. The increasing clutch age at which females of all three species switch from laying contiguously to noncontiguously parallels the development times of eggs in the three species, increasing from four days in S. partitus to 12-23 days in

86 COPEIA, 1995, NO. 1

H,rubicundus, and may reflect the decreasing difference in the developmental stage of em- bryos between consecutive days as development time increases.

Although we do not know of any other studies describing within-nest spawning-site prefer- ences in fishes, anecdotal information from re- searchers working on other damselfish species suggests that there may be substantial differ- ences among pomacentrids in their within-nest preferences. In the dusky damselfish, S. dorso-punicans, clutch placement is apparently not in- fluenced by the ages of other clutches present in a nest (C. W. Petersen, pers. comm.). In the orange-tailed blue damselfish, Chrjsiptera cj- anea, females prefer to deposit clutches among other clutches, but their position is not influ- enced by the ages of clutches present (RAK, pers. obs.).

There are several potential explanations for the within-nest spawning patterns shown by S. partitus, S. leucostictus, and H. rubicundus. First, females may prefer to lay contiguously to early- stage clutches to reduce the risk of per capita egg predation through a dilution effect (Roh- wer, 1978; Ridley and Rechten, 1981). Under this hypothesis, females should always lay con- tiguously to clutches already present in nests, regardless of their ages. Instead, females of all three species lay contiguously only to young clutches and lay noncontiguously to older clutches. Therefore, the dilution hypothesis does not appear to provide an explanation for the observed egg-laying patterns.

Second, female within-nest spawning-site preferences may result from different meta-bolic requirements of clutches as a function of clutch age. For example, late-stage clutches may have higher oxygen consumption rates than early-stage clutches (Alderdice et a]., 1958). If oxygen consumption is high enough, late-stage clutches may lower the amount of oxygen avail- able to nearby early-stage clutches, and reduced oxygen supply could cause developmental ab- normalities or retarded development. Under this hypothesis, females should lay contiguously to early-stage clutches and noncontiguously to late- stage clutches. Although the observed egg-lay- ing patterns are consistent with this hypothesis, nests of all three study species are open to ocean currents, making oxygen deprivation for entire clutches unlikely. Eggs at the junction of two clutches, however, could experience lower ox- ygen concentrations.

Third, within-nest egg-laying preferences by females in this study may be a consequence of a between-nest preference for early-stage

clutches. Female H. rubtcundus use the presence of early-stage clutches to discriminatk among potential nests (Sikkel, 1989) and our study in- dicates S. leucostictus females may show a similar preference. If females have a search image for early-stage eggs, they may be attracted to them within a nest and deposit their clutches nearby. When early-stage eggs are not present, females may simply deposit their clutches in any avail- able nest space. Although this hypothesis may explain the egg-laying patterns in H, rubzcundus and S. leucostzctus, our data suggest that S. par-tztus females do not use the presence of early- stage eggs to discriminate among potential nests but still have the same within-nest spawning-site preferences. Therefore, this hypothesis does not appear to be general enough to explain the very similar spawning-site preferences observed in all three study species.

Fourth, the within-nest spawning-site pref- erences may be a response to patterns of male parental care. In all three study species, males appear to direct more parental attention to younger clutches. This is particularly obvious in H, rubicundus, where males fan young clutch- es with the entire lower portion of the body but fan older clutches with pectoral fins only (Sik- kel, 1989). In all three species, young clutches have a much cleaner appearance than old clutches. If increased male parental care is im- portant to the survival of young clutches, fe- males may be able to increase offspring survival by laying among clutches in a similar develop- mental stage. In nests containing only old clutches, clutches may receive more parental care if they are laid noncontiguously. There- fore, egg-laying patterns of all three species are consistent with the "differential care" hypoth- esis, and this hypothesis warrants further study.

Despite the strong within-nest spawning-site preferences observed in all three study species, we were unable to detect any benefit in terms of clutch survival as a consequence of these pref- erences in S. partitus. Differences in clutch sur- vival are expected under the differential care hypothesis. There are two potential shortcom- ings of our clutch survival data that may have prevented us from detecting survival differ- ences. First, to determine whether female with- in-nest spawning-site preferences had conse-quences for clutch survival, we compared the survival of those clutches laid in the "wrong" position with the survival of clutches laid in the "correct" position. However, it is possible that, at the time a clutch was laid in the "wrong" position, this may in fact have been the best available location. If this was the case, no dif-

KNAPP ET AL.-CLUTCH DEPOSITION PATTERNS IN DAMSELFISH 8 7

ference in survival between clutches laid in the "correct" or "wrong" position would be ex- pected.

Second, our test of whether female within- nest spawning-site preferences result in in-creased clutch survival used survival data for the first day a clutch was present in the nest instead of for the four days between laying and hatching. This was necessary because the de- position of subsequent clutches contiguous to each test clutch would confound any effects of clutch position on egg survival. However, any negative effects on clutch survival caused by fe- males laying clutches in the "wrong" position may only be detectable over the full four-day developmental period. In addition, although we hypothesized that within-nest spawning site preferences would have consequences for egg survival, we cannot eliminate the possibility that clutches laid in the "wrong" position suffer sub- lethal effects.

Although we were unable to detect any con- sequence of female within-nest spawning-site preferences on female fitness, a recent study by Sikkel(1994) on H. rubicundus showed that these preferences have important consequences for male fitness. As a result of the female prefer- ence to lay initial clutches (i.e., the first clutch in a nest) near the nest periphery and the sub- sequent female preference to lay contiguously to early-stage clutches, nests generally accu-mulate clutches from one side to the other. However, females occasionally lay initial clutch- es in the center of the nest. In these cases, nests accumulate clutches until the nest boundary is reached; but because females strongly prefer to lay contiguously to early-stage clutches and avoid nests with only old clutches exposed to available nest space, these nests often remain half empty. In response, males often cannibalize the older clutches to expose the younger clutches to avail- able nest space. Consequently, females begin spawning in such nests again, and nests often accumulate clutches until all available nest space is occupied. Based on these results, Sikkel(1994) concluded that filial cannibalism in the garibaldi is an adaptive response to female within-nest spawning-site preferences.

RAK was supported by grants from the Na- tional Science Foundation (Dissertation Im-provement Grant BSR 91 1 1546), American So- ciety of Ichthyologists and Herpetologists (Raney Award), Lerner-Gray Fund for Marine Research, and Sigma Xi. PCS was supported by

grants from the American Society of Ichthy- ologists and Herpetologists (Raney Award), Lerner-Gray Fund for Marine Research, Sigma Xi, and the Drown Foundation. We thank D. Chiller, E. Graham, J. Kovach, D. Lirman, L. Mogran M. Steele, and E. Sturm for their ex- cellent field assistance during this research. R. Warner and E. Schultz made several suggestions that greatly improved the manuscript.

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(RAK) SIERRA NEVADA AQUATIC RESEARCH LABORATORY, OFUNIVERSITY CALIFORNIA, STAR ROUTE 1 , BOX 198, MAMMOTH LAKES, CALIFORNIA SCIENCE93546; (PCS) MACLEAN CENTER,UNIVERSITY ISLANDS,OF THE VIRGIN JOHN BREWERS UNITEDBAY, SAINT THOMAS,

STATES VIRGIN ISLANDS 00802; AND (VTV)

CALIFORNIA, CALIFORNIASANTA BARBARA, 93106. Send reprint requests to RAK. Sub- mitted: 14 Feb. 1994. Accepted: 25 April 1994. Section editor: S. T. Ross.