Behavioral Ecology Advance Access originally published online on July 20, 2005
Behavioral Ecology 2005 16(5):914-921; doi:10.1093/beheco/ari076
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Paternity and paternal effort in the pumpkinseed sunfish
Department of Biological Sciences, State University of New York at Buffalo, Buffalo NY 14260, USA
Address correspondence to O. Rios-Cardenas, who is now at the Department of Biological Sciences, Ohio University, Athens, OH 45701, USA. E-mail: rios-car{at}ohio.edu.
Received 4 February 2005; revised 22 May 2005; accepted 29 May 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Theoretical models suggest that males should adjust their parental effort according to paternity when parental effort is costly, paternity varies among clutches, and males have a cue to assess paternity. To date, nearly all tests of this theory have been conducted using birds as model organisms. In this study we examined these three factors and the relationship between paternity and male parental care in a fish system. In the pumpkinseed sunfish (Lepomis gibbosus), parental care is provided exclusively by males (parentals), but some males (sneakers) parasitize others by sneaking fertilizations. Parental males significantly lost weight during the parental care period. Clutch size and amount of parental effort did not affect a male's probability of obtaining more eggs. Paternity was variable among broods. The proportion of young sired by a parental male was not associated with frequency of fanning eggs or defense of hatched young, but was positively correlated with levels of nest defense during the egg stage. Egg survivorship might restrict an adjustment of fanning behavior, and a general decline in parental behavior (with brood age) might explain the lack of adjustment once the eggs hatch. Parental males did not adjust their care when we experimentally manipulated one possible cue of paternity. Together, these results indicate that male pumpkinseeds do adjust their care in relation to paternity, but the cues used to assess paternity are not clear.
Key words: alternative strategies, parental care, paternity, pumpkinseed sunfish.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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If paternal males can improve the probability of caring for their own genetic offspring by recognizing and favoring those young, or by abandoning broods in which they have sired few young, they can gain a selective advantage over indiscriminately paternal males (Westneat and Sherman, 1993
). Thus, we generally expect male parental effort (the expenditure of resources on parental care of offspring, Clutton-Brock, 1991) to be related to the proportion of a brood that the male has sired (i.e., "paternity," see Trivers, 1972).
Despite these arguments, the relationship between male parental care and paternity can be complicated by a number of factors. First, male parental care may be favored, even when the level of paternity is uniformly low, if a male improves the survival rate of those eggs he does fertilize enough to compensate for any resulting reduction in future reproduction (Werren et al., 1980). Second, the energetic costs of parental care may be offset by benefits in addition to improved offspring survival. For example, parental care may enhance future reproductive success by increasing the number of females visiting the nest (Unger and Sargent, 1988) or by increasing male social status (Wagner et al., 1996). Under these conditions male parental investment may be favored even if the brood contains a large proportion of young not related to the parental male.
Theoretical models describe the specific conditions under which males should facultatively adjust their parental effort in response to level of paternity (Houston, 1995; Kokko, 1999; Mauck et al., 1999; Werren et al., 1980; Westneat and Sherman, 1993; Whittingham et al., 1992; Winkler, 1987; Xia, 1992). These models suggest that the effect of paternity on parental effort should depend on three main factors (Westneat and Sherman, 1993): (1) whether parental care is costly (in the sense that it reduces alternative or future reproductive benefits), (2) whether paternity differs predictably between breeding attempts, and (3) whether males have reliable cues by which they can assess their paternity. To understand the relationship between male parental effort and paternity, studies must consider all of these key factors because the absence of any will likely prevent parental adjustment in response to reduced paternity (Westneat and Sherman, 1993; Whittingham et al., 1992).
Empirical research examining the relationship between male parental care and paternity has focused primarily on birds. Several such studies have reported a correlation between paternity and parental effort (e.g., Burke et al., 1989; Chuang-Dobbs et al., 2001; Dixon et al., 1994; Weatherhead et al., 1995), but others have not found such correlation (e.g., Gavin and Bollinger, 1985; Jamieson et al., 1994; Lifjeld et al., 1993; Morton et al., 1990; Stuchbury et al., 1994; Wagner et al., 1996; Westneat, 1988, 1995; Whittingham and Lifjeld, 1995). Such correlational studies are sometimes difficult to interpret because several factors may obscure relationships between paternity and paternal care. Specifically, variables such as male condition, timing of reproduction or male attractiveness may be confounded if they affect both paternity and paternal care (Kempenaers and Sheldon, 1997; Lifjeld et al., 1998a).
Some studies have examined paternity and parental care by experimentally manipulating perceived paternity (for reviews see Neff and Sherman, 2002; Westneat and Sargent, 1996; Wright, 1998). These studies have provided mixed results, with some studies showing the predicted relationship (e.g., Hartley et al., 1995; Koenig, 1989; Lifjeld et al., 1998b; Møller, 1988; Osorio-Beristain and Drummond, 2001; Sheldon and Ellegren, 1998; Sheldon et al., 1997; Wright and Cotton, 1994) and others not finding it (e.g., Birks, 1997; Dickinson, 2003; Kempenaers et al., 1998; MacDougall-Shackleton and Robertson, 1998; Westneat et al., 1995; Whittingham et al., 1993). However, most of these studies did not examine all three of the factors listed above; therefore, it is not clear whether males adjust their parental care when they are predicted to do so.
Male parental care is well developed in many other taxa, some of which are well suited to observational and experimental studies (e.g., Buchan et al., 2003; Hunt and Simmons, 2002). In particular, male parental care is well developed in fish; for example, in many species, males but not females provide parental care, and paternity is highly variable within and across broods due to the presence of "sneaker" males who parasitize the parental efforts of other males. Moreover, it is relatively easy to manipulate actual and perceived paternity of parental male fish (see Neff, 2003; Neff and Gross, 2001; Östlund-Nilsson, 2002; Svensson et al., 1998). Despite these advantages, few studies have examined the influence of paternity on male parental care in fish (but see previous references).
In this study we examined the relationship between male parental care and paternity in the pumpkinseed sunfish (Lepomis gibbosus). We used pumpkinseed sunfish because they present parental care by males, but not females, specialized sneaker males that parasitize the parental efforts of parental males, and potentially reliable cues of paternity (e.g., intrusions by sneakers during spawning). We tested whether conditions in this species would favor adjustment of paternal care by males in response to paternity. Specifically, according to theoretical models (see above), males should adjust their parental care in response to paternity when parental effort is costly to males (in terms of current and/or future reproductive success), and there is variation in paternity between clutches. In addition, we tested whether males do adjust their parental care as predicted by quantifying male parental behaviors and relating these to levels of paternity as determined by molecular genetic analysis. Finally, we tested if the manipulation of a possible cue of paternity (the presence of sneakers near the nest) affected the levels of paternal care.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Study system
Sunfish are freshwater, iteroparous fish common in slow-moving warm waters, ponds, and lakes throughout North East North America (Carlander, 1977
). In some species, including pumpkinseed sunfish, relatively large and old males ("parental males") build nests to which they attract females for spawning. Nests are circular depressions of the substrate formed when parental males "clean" or "sweep" their territories (Carlander, 1977). Parental males remain at the nest after spawning, guard and fan the eggs, and guard the non–free-swimming fry ("wrigglers") without female assistance. The parental care period lasts about 9 days (Carlander, 1977; Rios-Cardenas O, personal observation), at the end of which some males clean an adjacent area near the nest for another breeding attempt (Rios-Cardenas O, personal observation). However, not all males are parental: relatively small and young but sexually mature males ("sneakers") adopt an alternative reproductive tactic in which they do not construct nests and instead intrude during spawnings at the nests of parental males to fertilize eggs (Gross, 1979; Rios-Cardenas, 2003). Although parental males attempt to defend their paternity by aggressively chasing sneakers detected near the nest, sneakers successfully intrude during spawnings (Rios-Cardenas, 2003). Thus, parental males often provide care to young that were sired by sneaker males, and this mixture of sneakers and parental males is thought to exist as a balanced polymorphism in which the two strategies have equal fitness at an evolutionary equilibrium (Rios-Cardenas, 2003).
General methods
We conducted this study in Lincoln Pond, situated in The Edmund Niles Huyck Preserve, southwest of Albany, New York, USA, during the 1999–2001 breeding seasons. This site is a 4-ha pond that has experienced minimal disturbance (e.g., fishing has not been allowed there for more than 80 years). Pumpkinseeds are the only sunfish species inhabiting this pond. Other common fish inhabiting the pond were largemouth bass Micropterus salmoides, chain pickerel Esox niger, golden shiners Notemigonus crysoleucas, and fathead minnows Pimephales promelas.
In the field (using diving mask and snorkel), we located, mapped, and collected microhabitat data for 55 active nests. In nests with eggs or wrigglers, or where mating was observed, we performed a 30-min focal observation to record frequency of fanning (for each nest with eggs) and frequency of defensive behaviors. We recorded the following defensive behaviors: opercular displays (fish faced intruder and spread opercula), charges (fish moved toward intruder but stopped once intruder fled), chases (fish moved toward intruder and followed when it fled), and bites (fish touched intruder with its mouth). The frequencies of defensive behaviors were positively correlated with each other (Rios-Cardenas, 2003), suggesting a unitary intraspecific aggression system (Colgan and Gross, 1977). Therefore, to simplify analyses, we used a single measure of defense (PC1) calculated from a factor analysis involving all defensive behaviors. For nest with eggs, we performed an additional 30-min observation period once the eggs hatched to obtain a measure of defense for the wriggler stage. For an independent set of 18 nests with offspring in the wriggler stage, we recorded a standardized measure of defensive behavior (see Neff, 2003) by presenting the parental male with one conspecific intruder in a jar (conspecifics are cannibalistic in this species). We placed the jar with the intruder just outside the "rim" of the nest for 15 min and recorded defensive behaviors directed toward the intruder in the jar. For each nest observed, once we finished all observation periods, we collected the wrigglers and preserved them and captured the parental male (with a dip net). We individually marked each fish with a Petersen disc tag made of color sequins and collected a tissue sample from a fin.
The fertilization window of eggs is very short (i.e., less than a minute, Bagenal and Braum, 1971); thus, any defensive behavior directed to intruders (including possible sneaker pumpkinseeds) that does not occur during a mating event can be considered as guarding the brood (rather than guarding the paternity of the parental male). In addition, a positive correlation between paternity and brood defense may exist if there is a correlation between intensity of fertility defense and intensity of brood defense (e.g., if males in good condition are able to defend both their fertility and brood more vigorously). To test this possibility, for an independent subset of nests we observed both the defensive behaviors that territorial males directed toward sneakers during mating and brood defense.
Previous analyses showed that brood age affects both fanning and defense effort (Rios-Cardenas, 2003). Therefore, we conducted analyses of paternity and parental effort independently for the two stages of the brood (eggs and wrigglers) and used brood age as a covariate in most analyses. If parental males use paternal effort as a courtship display during the mating period (e.g., to increase the number of females attracted and subsequently acquire more eggs), our measure of parental effort might be overestimated during the early stages of the brood (it would include not only parental but also mating effort when the parental and mating periods overlap, see Rios-Cardenas, 2003). Statistically controlling for the effects of brood age removes any effects that mating effort may have on parental effort during the early stages of the brood. We also included clutch size and timing of reproduction as covariates in our analyses of fanning effort because previous analyses showed that these factors affected fanning of eggs (Rios-Cardenas, 2003).
All response variables were normally distributed and showed homoscedasticity. For analyses where the same data set were used for different tests, we used Bonferroni corrections to adjust the critical 
levels accordingly (Sokal and Rohlf, 1995). The corrected critical levels (') are indicated along with the statistics for such tests.
Effect of parental effort on reproductive success
To assess effects of parental effort on future reproductive success, we examined whether male parental care is energetically costly in this species by measuring the weight (to the nearest 0.1 g with a digital scale) of 27 parental males captured at two different points during the parental period: immediately after receiving the first batch of eggs, and again just before the free-swimming fry dispersed (4 days after hatching, 7 days after receiving the eggs). In addition, in 18 of these fish (where behavioral data were available) we tested for a correlation between weight loss and amount of parental effort.
The energetic costs of parental care might be mitigated if males with eggs in their nest are more likely to receive eggs from additional females than are males without eggs, as has been found in some other fish (Unger and Sargent, 1988). We investigated this possibility in two ways. First, we tested for an effect of clutch size on spawning success by monitoring an independent set of 85 unmanipulated nests. Because males are "receptive" to females and will accept additional eggs only during the first 2 days of egg tending (Gross, 1980; Rios-Cardenas O, personal observation), we measured the size of the clutch in each of these nests for the first and second day of the parental care period to determine if they received additional eggs. We measured clutch area by placing a plastic hoop of known perimeter (area inside the hoop = 2922.5 cm2) over the clutch and estimating the percentage of the area inside the hoop covered by eggs. Second, we experimentally manipulated the clutch size of an independent set of 23 nests. For this experiment, prior to spawning we covered clean nests with several letter-sized sheets of transparent-dull plastic. After eggs were laid we could manipulate clutch size by moving the plastic sheets. We randomly assigned each nest to one of the four treatments: (1) full clutch removal (n = 6), (2) half clutch removal (n = 5), (3) manipulated control (manipulating the sheets without removing any eggs, n = 5), or (4) clutch size increase (in which clutch size was increased approximately 50% by adding eggs from one of the removal nests, n = 7). We then assessed whether experimental manipulation affected the probability that a nest would receive additional eggs (as indicated by changes in clutch area after manipulation).
Energetic costs of parental care might also be mitigated if parents who provide high levels of care receive more eggs. To test this possibility, we considered a subset of the unmanipulated nests for which clutch size had been obtained as indicated above, and for which parental behaviors were measured during the day they received the first eggs (N = 18) to analyze the relation between rates of care of parental males and the probability that their egg clutches would increase in size (evaluated as described above).
Finally, to estimate the effects of fanning behavior on survival of the eggs, we tested for a correlation between nesting success and frequency and duration of the fanning behavior. To estimate nesting success we first measured clutch area for an independent set of 16 nests and used a linear regression equation previously obtained (linear regression: R = .97, F1,6 = 82.89, N = 8, p < .001, for clutch area and number of eggs) to estimate the number of eggs in those nests. Second, we collected all the wrigglers in those nests the day they hatched (usually when they are 4 days old). In the laboratory, we obtained those wrigglers' dry weight and used a linear regression equation previously obtained (linear regression: R = .99, F1,3 = 127.13, N = 5, p < .01, for wriggler dry weight and number of wrigglers) to estimate the number of wrigglers in those nests. We finally calculated nesting success by dividing the number of wrigglers hatched by the number of eggs originally obtained. Note that the definition of nesting success includes the effects of both nest predation (loss of eggs before wrigglers were collected) and hatching success (the per-egg probability of hatching). We performed a 15-min focal observation (when eggs were 2 days old) to record frequency and duration of fanning behavior for these nests.
Paternity analyses
We used five microsatellite markers previously isolated from bluegill (Lma25, Lma57, and Lma87, Colbourne et al., 1996) and redbreast sunfish (RB7 and RB20, DeWoody et al., 1998) to assess paternity at 55 nests. For each of these nests, we genotyped a random subsample of wrigglers (average = 49 wrigglers per nest, range = 42–50). We also genotyped 174 adults, including the parental male at each nest and the other possible putative parents that we had sampled. We used the adult genotypes (which included 21 females and 20 sneakers) to calculate allele frequencies in the breeding population.
We amplified genomic DNA from each individual in two 10-µl multiplex polymerase chain reactions (PCRs). One reaction (M2) used the Lma87 and RB20 primers, while another (M3) used the Lma25, Lma57, and RB20 primers. These loci combinations were selected because preliminary analyses showed that the distributions of allele sizes did not overlap within each multiplex set. For labeling purposes, all forward primers ended with the M13 forward sequence. Both reactions contained (final concentrations) 0.10 mg/ml bovine serum albumin, 0.60 mM deoxynucleotide triphosphate mix, 1x PCR buffer, 0.10 µM fluorescent dye–labeled M13 forward primer (Li-Cor Incorporated, Lincoln, NE), and 1 unit Taq DNA polymerase. The M2 reaction contained 0.01 µM RB20 primers (each), 0.10 µM Lma87 primers (each), and 2.50 mM MgCl2. The M3 reaction contained 0.01 µM Lma25 and Lma57 primers (each), 0.10 µM RB7 primers (each), and 3 mM MgCl2. Both reactions were subject to 30 cycles of 94°C for 60 s, X°C for 60 s, and 72°C for 45 s, where the annealing temperature (X) was 60°C for M2 and 57°C for M3. Amplification products were then size sorted by electrophoresis in a 5.5% denaturing polyacrylamide gel containing 7 M urea at a constant temperature of 50°C and read by an automated sequencer (Li-Cor NEN Global IR2 DNA Analyzer System).
We used the GeneImagIR software (Scanalytics Incorporated, Fairfax, VA) to score the size of PCR fragments for each individual by comparing its band(s) to a size standard (Li-Cor Incorporated) For each nest analyzed, we compared offspring genotypes to those of the parental (nest) male to determine the proportion of sampled young that he had sired. The male was excluded as the sire of an offspring if he did not match at one or more loci.
To estimate the proportion of paternity for each parental male we used the two-sex paternity model presented in Neff et al. (2000a). We selected this model because multiple females and multiple males can spawn in each pumpkinseed nest. This formula uses the parental multilocus genotype, the genotypes of the brood in his nest and allele frequencies in the breeding population (to consider the probability that a wriggler could match by chance). We used exact tests in GENEPOP (Raymond and Rousset, 1995) to test if allele frequencies at each locus deviated from Hardy-Weinberg expectations. The exclusion probability (defined as 1 – NGdad, where NGdad is the expected proportion of next generation individuals that is compatible with the putative father by chance; for calculation see Neff et al., 2000a) considers all loci used in the analysis and is calculated independently for each nest (see Neff et al., 2000b). We calculated a mean exclusion probability by averaging the probabilities for individual nests.
Cues to assess paternity
We tested one possible cue of paternity—the presence of sneaker males near a nest—in an experiment that manipulated this cue (see also Neff, 2003). Specifically, we randomly selected an independent set of 22 clean nests (where the male was ready to receive eggs) and placed two jars near each of these (just outside the rim of the nest). At half of the nests (experimentals) each jar contained a sneaker male, and at the other half (controls) the jars were empty. Because we placed the jars prior to spawning and removed them 1 day after parental males received their first eggs, they remained at the nest for 2–3 consecutive days. We observed the parental males immediately after the removal of the jars (when their eggs were 2 days old) to obtain the measures of parental effort (as described above). We then compared the parental behavior of experimental males to that of controls.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Effect of parental effort on reproductive success
Parental males significantly lost weight during brood care (between the day they received eggs and just before their brood dispersed; paired t test: t26 = 5.67, N = 27,
' = 0.017 after Bonferroni correction, p < .001). The percent weight loss was on average 6.3%. However, the amount of weight lost was not significantly correlated to the amount of parental effort males provided [correlation: r = .236, N = 8, p = .57, 95% confidence interval (CI) for the effect size = –0.585 to 0.794 for fanning effort; r = –.017, N = 18, p = .95, 95% CI for the effect size –0.454 to 0.479 for defensive behaviors (PC1)].
The analysis of unmanipulated clutches showed that having a large clutch during the first day of egg tending did not improve the probability of subsequently receiving more eggs; on the contrary, small clutches were significantly more likely to increase in size and large clutches were significantly more likely to decrease in size for the second day of egg tending (log likelihood ratio test: 
N = 85, ' = 0.017 after Bonferroni correction, p < .001). Furthermore, the clutch manipulation experiment showed that nests where eggs were removed subsequently acquired more eggs, whereas control nests and those where eggs were added tended to lose eggs (ANOVA: F3,19 = 6.134, N = 23, p < .01, Figure 1).
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The amount of parental effort provided to the clutch did not improve the probability of subsequently receiving more eggs (log likelihood ratio test:
N = 18, p = .49 for fanning). Indeed, clutches of males that provided higher levels of defense (PC1) were more likely to remain unchanged rather than to increase in size. However, this same analysis also showed that clutches of males that provided lower levels of defense were significantly more likely to decrease (log likelihood ratio test: 
N = 18, ' = 0.017 after Bonferroni correction, p < .01), suggesting that high levels of defense decrease the probability of predation. Finally, nesting success was not affected by fanning frequency (correlation: r = .06, N = 16, p = .84, 95% CI for the effect size: –0.45 to 0.54) or time spent fanning (correlation: r = –.13, N = 16, p = .62, 95% CI for the effect size: –0.58 to 0.39).
Variability of paternity among broods
The microsatellite loci used were highly variable with each having from 4 to 12 alleles and heterozygosities ranging from 0.54 to 0.84. In all cases, genotype frequencies did not differ significantly from Hardy-Weinberg expectations (all p > .06), suggesting that null alleles were not prevalent. The average exclusion probability (using all five loci) indicated a high probability (0.90) of excluding the parental male if he was not the true sire and the model accounts for the remaining 10% (see Neff et al., 2000b).
Paternity was variable among independent broods, ranging from a minimum of 0.43 to a maximum of 1 (
N = 55). Thus, parental males do lose paternity to sneakers and often provide care to young that are not their own.
Parental effort and paternity
To analyze if males adjust their parental effort in response to paternity, we examined the egg stage, when males fan and defend the young, separately from the wriggler stage, when males defend but do not fan the young.
After controlling for brood age (a), brood size (s), and time of reproduction (t), there was no significant correlation between paternity (p) and the frequency of fanning eggs (f) (partial correlation: rpf.ast = –.17, N = 33, p = .37). However, during the egg stage, the intensity of male nest defense (PC1) was positively associated with paternity (partial correlation: rp(PC1).a = .409, N = 33, ' = 0.025 after Bonferroni correction, p = .02, Figure 2; note that out of the 55 parental males sampled, only 33 were observed during the egg stage). In addition, rates of defensive behaviors against sneakers during mating were not related to rates of nest defense (linear regression: R = .13, F1,33 = 0.55, N = 35, p = .46, 95% CI for the effect size: –0.22 to 0.44).
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When using behavioral data collected during the wriggler stage, neither natural nor standardized (using intruder in jar) defensive behaviors were significantly correlated to paternity (partial correlation: rp(PC1).a = .16, N = 52, p = .25 for natural defense; and rp(PC1).a = –.05, N = 18, p = .84 for standardized defense).
Cues to assess paternity
The constant presence of two sneakers in jars around the nest during mating did not affect the subsequent parental behavior of experimental males relative to controls [ANOVA: F1,20 = 0.072, N = 22, p = .79, effect size: r (95% CI) = .059 (–.374 to .467) for fanning; and F1,20 = 0.059, N = 22, p = .81, effect size: r (95% CI) = .054 (–.378 to .463) for defensive behaviors (PC1)].
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Theoretical models suggest that, if reliable cues of paternity are present, males should reduce their parental care when care is costly and paternity varies across clutches (Sheldon, 2002
). Our results suggest that all of these conditions hold for pumpkinseed sunfish. First, even though we did not monitor weight loss of juvenile or nonreproductive but mature individuals, weight loss during the parental care period suggests that parental effort in this species is energetically costly (but see below). Second, paternity is clearly variable among broods of pumpkinseed sunfish. Because parental effort is costly, we would expect parental males to reduce this cost whenever the benefits of parental care are low (e.g., when paternity is low). This expectation was met in this study, but only for nest defense during the egg stage.
Pumpkinseed sunfish do not feed during the winter, as the stomach is shrunken and mucus filled, but an increase in water temperature initiates a feeding response (Carlander, 1977). Furthermore, growth rate (measured as weight gain) and average food intake increase with water temperature, particularly during the breeding season months (May–September; Carlander, 1977). Therefore, parental males seem to lose weight in large part because they do not leave the nest to feed during the parental care period. This reduced feeding while tending the nest is directly related to providing care to the brood. In fact, the only individual that gained weight during this period was observed eating a large crayfish that had recently molted inside his territory. This energetic cost likely affects future survival and/or reproductive success because males suffer heavy mortality at the end of the breeding season (Gross, 1980). Furthermore, our observational and experimental evidence shows that the energetic cost of parental care is not mitigated by additional matings because neither the size or presence of an egg clutch nor the amount of parental effort increased a parental male's ability to obtain additional eggs.
Because we did not find a direct relation between the variation of defensive effort (the variable that was positively related to paternity) and the variation of weight loss during the parental care period, defensive effort might be considered as not costly. However, even if defensive effort is not the main cause of weight loss, parental defense activities certainly contribute to the deficit of energy revealed by this weigh loss. Furthermore, we suggest parental effort has other costs that were not examined in our study (e.g., increased risks of predation and of agonistic retaliation and subsequent aggressive escalation by the intruders; see Grant, 1997 for a review).
For males to adjust their care, they must have a reliable cue of paternity. Our results indicate that male pumpkinseed sunfish do not use the presence of sneakers around the nest as such a cue. Although the 95% CI of the effect size appear to be large for these experiments (see Colegrave and Ruxton, 2003; Jennions and Møller, 2003), even the largest estimates of the effect size within these 95% CI (0.47 for fanning and 0.46 for defensive behavior) would correspond to nonsignificant F values (F1,20 = 5.58 for fanning and F1,20 = 5.32 for defensive behaviors, which are smaller than the critical value F0.05(2),1,20 = 5.87) (for conversions between test statistic and effect size see Rosenthal, 1994).
Using a similar experimental approach to manipulate perceived paternity, Neff (2003) found that bluegill sunfish (Lepomis macrochirus) responded to the presence of sneakers around nests by decreasing their care. The lack of response by pumpkinseed males to this same cue might be explained by differences in nest density and spawning synchrony between pumpkinseed and bluegill sunfish. In dispersed nesters like pumpkinseeds, females spawn for longer periods and spawnings do not tend to occur simultaneously at different nests. Thus, pumpkinseed parental males can better defend their spawnings (Gross, 1982), and the sneakers present near nests might not necessarily intrude or fertilize eggs. Therefore, in the pumpkinseed sunfish the actual number of intrusions during spawnings may be a more reliable cue of paternity than the mere presence of sneakers near the nest. Unfortunately, we were unable to manipulate this potential cue, and we were also unable to test for a correlation between intrusions during spawning and paternity due to a low number of sampled nests where matings were observed. However, an inverse relation between sneaker intrusion rates and paternity has been described in the closely related bluegill sunfish (Fu et al., 2001). If this relation is true also for pumpkinseed sunfish, males may be using actual intrusion rates, rather than the presence of sneakers, as a cue by which to estimate their paternity.
Although pumpkinseed males adjusted their defense of eggs, they did not adjust their fanning of eggs in relation to paternity. There are three possible explanations that might account for this difference. First, besides being important for egg survival, fanning may also speed up the development of eggs (e.g., Sargent, 1985), such that males may not decrease their fanning if this prolongs the parental period. However, there was little variation in the duration of the egg stage (3 days), and changes in the duration of this stage seemed to be associated with severe changes in water temperature, rather than with variation in fanning rates (Rios-Cardenas O, personal observation). Second, if reducing fanning below a certain level (threshold) causes very high mortality, adjustment of fanning behavior might be constrained by egg survival, such that minor adjustments (above the threshold) will not affect nesting success significantly. Although variation in fanning effort seems to have little effect on nesting success (this study, Gross, 1980), clutches that are not fanned at all suffer high mortality (Gross, 1980). In contrast, levels of defensive behavior do appear to affect brood survival, as clutches with low levels of defense were more likely to decrease in size (see also Gross, 1980). Pumpkinseed sunfish males might therefore be adjusting only those behaviors (e.g., defensive effort) for which variation does affect brood survival.
Third, because weight loss was not significantly correlated to fanning rate, pumpkinseed males might not be able to reduce energetic costs with minor adjustments of fanning behavior. Our results suggest that variation of fanning effort is not a major cause of variation of weight loss during the parental care period. However, the sample size used to analyze the correlation between weight loss and fanning behavior was small and, as indicated by the wide 95% CI of the effect size (Colegrave and Ruxton, 2003; Jennions and Møller, 2003), the test might not have had enough statistical power to detect a correlation between these variables. Further tests with larger sample sizes are necessary to reliably test if rates of parental behavior have a significant effect on male weight loss. In contrast, defensive effort may be both energetically costly and risky in terms of the danger of exposure to predators and agonistic retaliations (see above), thus pumpkinseed males might be able to substantially reduce these costs by adjusting defensive behaviors according to paternity.
To our knowledge there have been only four other studies analyzing the effects of paternity on parental effort using fish. Two of these studies (Svensson et al., 1998, with the common goby; and Östlund-Nilsson, 2002, with 15-spined sticklebacks) showed a lack of adjustment of parental effort to reduced paternity (estimated by the presence or absence of sneaking attempts between treatment groups). In both studies, the authors suggest that the lack of response may have been caused by the inability of males to assess their paternity. However, in both cases the actual variation of paternity between clutches in the different treatments was not established. If levels of paternity are similar between clutches (independent of the frequency of sneaking attempts), fathers are not expected to adjust the amount of effort provided (Westneat and Sherman, 1993). This scenario would have occurred in the described studies if sneaking attempts do not always result in fertilizations. Therefore, a correlation between variation of paternity and levels of sneaking attempts (their proposed cue of paternity) should be confirmed in these species.
The other studies (Neff, 2003; Neff and Gross, 2001) found that male bluegill sunfish facultatively adjusted their parental care in response to paternity cues. Moreover, this response was dynamic: during the egg stage males adjusted effort based on the frequency of sneakers during mating (Neff and Gross, 2001), and during the wriggler stage males further adjusted their care based on actual paternity (assessed by an olfactory cue, Neff and Sherman, 2003). Because brood defense in bluegill sunfish is higher during the wriggler stage than during the egg stage (Neff and Gross, 2001), further adjustment of parental care may be beneficial. In contrast, in pumpkinseed sunfish there is a large drop in overall parental effort during the wriggler stage (Rios-Cardenas, 2003) as males prepare for additional reproductive bouts (some males even start cleaning new nests during the last days of the parental care period). Thus, the small amount of care (including defense) provided during the wriggler stage may leave little room for behavioral adjustment and may make an adjustment during the wriggler stage unnecessary.
The evidence for an adjustment of parental effort to reduce paternity presented in this study is correlational. However, because rates of fertility defense were not related to rates of brood defense, the reduction of nest defense during the parental care period according to paternity does not appear to be related to an intrinsic defense capacity (e.g., condition) of parental males. Furthermore, timing of reproduction, another possible confounding variable, only affected fanning behavior (Rios-Cardenas, 2003) and was considered in analyses that involved that variable. Finally, male attractiveness might be a confounding variable, especially if some of the parental effort provided also serves as mating effort (e.g., Pampoulie et al., 2004). For the pumpkinseed sunfish this might occur during the first 2 days of the parental care period (when males are still accepting additional eggs). We statistically controlled for differences in parental effort during periods when mating effort might have existed by considering brood age as a covariable in all analyses. In addition, the amount of eggs obtained was not related to either parental care (this study) or paternity (Rios-Cardenas, 2003). Finally, levels of defense during the first 2 days of the parental care period affect the number of young produced, but not the number of mates attracted (Rios-Cardenas, 2005). Therefore, mating effort does not appear to be a confounding variable in this case. Nevertheless, experimental studies that manipulate cues of paternity would definitively demonstrate that male pumpkinseed sunfish adjust their parental care in response to paternity, as has been shown for bluegill sunfish (Neff, 2003).
This and similar studies (e.g., Neff, 2003; Neff and Gross, 2001) open the door to experimental approaches that more specifically test the variables affecting the relationship between parental effort and paternity. For example, one could experimentally test for the relationship between sneaking rates and male parental care by manipulating the density of sneakers in a pond or by manipulating population density (e.g., Hunt and Simmons, 2002) to examine its effects on sneaking rates and paternity. Such experiments could establish a causal relationship between sneaking rates and parental care.
Kempenaers and Sheldon (1997) suggested that, in general, studies of paternity and parental effort in birds show a lack of relationship between these two variables (see also Kempenaers and Sheldon, 1998; Lifjeld et al., 1998a; Wagner et al., 1998). The difficulty of identifying paternity cues to experimentally manipulate in bird studies may reflect a problem that birds could be having themselves: a lack of reliable paternity cues. Sunfish do not seem to be facing the same problem, as sneaking events should be easily perceived and reliably signal paternity of the males being cuckolded. This may be the reason why this and other studies (Neff, 2003; Neff and Gross, 2001) using sunfish as a model to examine the relationship between parental effort and paternity have yielded positive results. Thus, facultative adjustment of parental care in response to reduced paternity may be more common in fish than in birds or other taxa that lack reliable paternity cues. Alternatively, processes that significantly affect paternity predisposition (Neff and Sherman, 2002) may be more common in birds, and future studies should consider this factor when studying the effect of paternity on parental care.
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ACKNOWLEDGEMENTS |
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We would like to thank Mary Alice Coffroth, Derek Taylor, and Carol Berman for comments that greatly improved this research. Goran Arnqvist and two anonymous reviewers also provided helpful comments. We are grateful to The Edmund Niles Huyck Preserve and Biological Field Station and Guiyun Yan for logistic support provided during the course of this study in the field and in the laboratory, respectively. Michelle Rainka provided invaluable help in the field and in the laboratory. We are grateful to Samuel Holzman and Victor Callirgos for assistance in the field, and Nancy Urban, Dmitriy Akselrod, and Christina Costa for assistance in the laboratory. O.R.-C. was supported by an IIE/Fulbright/Garcia-Robles/CONACYT fellowship during this study. This study was supported by grants from the Graduate Group in Evolutionary Biology and Ecology at SUNY Buffalo, Graduate and Post Graduate Research Grants from the E. N. Huyck Preserve and a Doctoral Dissertation Improvement grant from the National Science Foundation (IBN-0073536).
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FOOTNOTES |
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M. S. Webster is now at the School of Biological Sciences, Science 312, P.O. Box 644236, Pullman, WA 99164-4236, USA
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