This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Animal Behaviour 78 (2009) 747–753 Contents lists available at ScienceDirect Animal Behaviour journal homepage: www.elsevier.com/locate/yanbe Potential reproductive rate of a sex-role reversed pipeﬁsh over several bouts of mating Sunny K. Scobell*, Adam M. Fudickar 1, Rosemary Knapp 2 Department of Zoology, University of Oklahoma a r t i c l e i n f o Article history: Received 5 February 2009 Initial acceptance 19 March 2009 Final acceptance 28 May 2009 Published online 4 August 2009 MS. number: A09-00073 Keywords: gulf pipeﬁsh life-history theory ovarian morphology pipeﬁsh potential reproductive rate sex-role reversal sexual selection Syngnathidae Syngnathus scovelli The potential reproductive rate (PRR, the rate at which each sex could reproduce if given unlimited mates) has proven to be a useful tool in predicting the direction and strength of sexual selection. We conducted a 2-month study of the PRR in the polyandrous gulf pipeﬁsh, Syngnathus scovelli, a year-round breeder. In this sex-role reversed species, the female transfers eggs to a male’s brood pouch during mating and thus renders him unavailable to mate for 2 weeks. We predicted females would have a higher PRR than males and that the rates in both sexes would change over successive breeding bouts in relationship to previous reproductive output. Females did have a higher overall PRR than males for the entire study period. However, PRR was not constant across individual breeding bouts. For each sex, the PRRs from the ﬁrst and third bouts of mating were signiﬁcantly higher than the PRR of second mating bout. Our results are consistent with individuals making trade-offs between current and future reproductive investment. We also discuss how ovarian morphology may contribute to elevated female PRR in this species. To our knowledge, this is the ﬁrst study of PRR in a North American pipeﬁsh. The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. When studying mating behaviour, researchers usually want to know which sex is under greater sexual selection pressure and to what degree. This information is useful in determining the sex that should be competitive for mates and the sex that should be choosy. The strength of sexual selection acting on each sex can also be correlated with territoriality, the degree of sexual dimorphism and/ or ornamentation and variance in mating success (Andersson 1994). However, measuring the strength of sexual selection acting on each sex has been difﬁcult historically, and determining which methodology is best has been the subject of debate. Many researchers have measured sex differences in parental investment and/or the operational sex ratio, two indirect measures of the strength of sexual selection. Trivers (1972) proposed that the sex that has relatively greater parental investment (PI) should be choosy, leaving the sex that has lower PI to compete for mates. * Correspondence and present address: S. K. Scobell, Department of Biology, Texas A&M University, 3258 TAMU, College Station, TX 77843-3258, U.S.A. E-mail address: [email protected] (S.K. Scobell). 1 A. M. Fudickar is now at the Department of Migration and Immuno-ecology, Max Planck Institute for Ornithology, Radolfzell, Germany. 2 R. Knapp is at the Department of Zoology, University of Oklahoma, 730 Van Vleet Oval, Room 314, Norman, OK 73019, U.S.A. However, PI (deﬁned as any effort that increases offspring survival at the expense of the parent’s ability to invest in other offspring) can be difﬁcult to measure, particularly in species where both sexes have some form of investment (Clutton-Brock 1991). Emlen & Oring (1977) proposed that a bias in the operational sex ratio (OSR) away from 1:1 could result in an increase in the intensity of sexual selection on the sex that is more abundant. The OSR, which is the average ratio of sexually active females to males in a population at any given time, has been effective in estimating the opportunity for sexual selection in several species (Vincent et al. 1994; Kvarnemo & Ahnesjö 1996; Dearborn et al. 2001; Jones et al. 2001). However, obtaining an accurate OSR for a population may not be feasible for a particular species being studied and could also vary greatly temporally (Forsgren et al. 2004), which could in turn have consequences for conclusions about the strength of sexual selection. In situations where it is not practical to estimate the PI or OSR, the potential reproductive rate can be used to predict the intensity of sexual selection. The potential reproductive rate (PRR) is the rate at which each sex in a population could reproduce if given unlimited mates (Clutton-Brock & Vincent 1991) and assumes no differential immigration or mortality between the sexes (Kokko & Monaghan 2001). The PRR has proven to be a useful tool in predicting the direction and strength of sexual selection (ﬁsh: 0003-3472/$38.00 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. doi:10.1016/j.anbehav.2009.05.036 Author's personal copy 748 S.K. Scobell et al. / Animal Behaviour 78 (2009) 747–753 Kvarnemo 1994; Kvarnemo & Ahnesjö 1996; Masonjones & Lewis 2000; Ahnesjö et al. 2001; Wilson 2009; insects: Kvarnemo & Simmons 1998; Wiklund et al. 1998; mammals: Preston et al. 2005). It is usually measured under controlled laboratory conditions where competition for mates is eliminated and environmental variables are kept constant (i.e. temperature, food, shelter, lack of predation, etc.). Thus, the PRR controls for many of the confounding variables that can inﬂuence reproductive function and gives an estimate of the baseline reproductive output for each sex under a similar, constant environment. PRR is typically measured in one of two ways: the number of offspring (or potential offspring) produced per unit time (CluttonBrock & Vincent 1991; Ahnesjö et al. 2001; Wilson 2009) or the ‘time in/time out’ model (Clutton-Brock & Parker 1992). CluttonBrock & Vincent (1991) deﬁned the PRR as the maximum number of offspring that each sex produced per unit time. Later, Ahnesjö et al. (2001) measured PRR by counting the number of ‘potential’ offspring produced per day over the course of the breeding season. They argued that the female’s PRR should be based on the number of eggs released during mating and that the male’s PRR should be based on the number of eggs fertilized. For the ‘time in/time out’ model, Clutton-Brock & Parker (1992) deﬁned ‘time out’ as the time period in which an adult is not able to mate, and they deﬁned ‘time in’ as the time period when an animal is able to mate if given a receptive partner. Both methods (‘time in/time out’ and number of offspring/unit time) have proven useful and which method is used often depends on the mating system of the species being studied. The PRR has been used to study the mating system of several species of syngnathids (pipeﬁsh and sea horses). The family Syngnathidae is unique among ﬁshes in that male pregnancy is found in all species and some, but not all species, are sex-role reversed (Vincent et al. 1992). In species where the PRR of females exceeds that of males, the mating system is typically sex-role reversed and females compete for access to males. Nerophis ophidion and Syngnathus typhle females were estimated to be able to ﬁll the pouch of one male completely and still have a 41% surplus of eggs remaining to give to another male (Berglund et al. 1989). Ahnesjö (1995) later calculated that S. typhle had a female-biased of PRR 1.8:1 (females to males) in warm water and 2.3:1 in cold water. Both N. ophidion and S. typhle are pipeﬁsh that have sex-role reversed mating systems in which females compete for access to males (Berglund & Rosenqvist 2003). In species where the PRR of males and females approaches unity, the mating system is typically monogamous with conventional sex roles (but see Sogabe & Yanagisawa 2007). Vincent et al. (1994) were the ﬁrst to show that despite their pregnancy, male Sri Lankan sea horses, Hippocampus fuscus, had a higher PRR and displayed more competitive and courtship behaviour than females. Similarly, Masonjones & Lewis (2000) found that dwarf sea horses, Hippocampus zosterae, have a PRR of 1:1.2 and also show a conventional, monogamous mating system. However, the PRR does not always predict a species’ mating behaviour. Sogabe & Yanagisawa (2007) studied the PRR of the monogamous messmate pipeﬁsh, Corythoichthys haematopterus, and found that there was no difference in PRR between the sexes, but females were more active in courtship than males. The observed behavioural differences were associated with a femalebiased OSR for this population. Further studies are needed in this family to determine how PRR is related to the mating system. We conducted a study of the PRR of the sex-role reversed gulf pipeﬁsh, Syngnathus scovelli, over a 2-month period. The gulf pipeﬁsh (Evermann & Kendall 1896) is a common syngnathid found throughout the Gulf of Mexico and breeds year round in Florida, U.S.A. (Reid 1954; Joseph 1957; Brown 1972). Gulf pipeﬁsh males, like many members of the family Syngnathidae, have a pouch into which the female deposits her eggs (her only known contribution). The male fertilizes the eggs and then broods them for about 14 days (Brown 1972). Thus, male gulf pipeﬁsh contribute signiﬁcantly more time to parental care than do females. The gulf pipeﬁsh is also sexually dimorphic and has a polyandrous mating system. Females are typically larger than males and have a sexually dimorphic ornament, a silver bar on each bony ring of the trunk (Reid 1954). Jones et al. (2001) found that mated females in the ﬁeld had a larger mean snout–vent length, body depth and body mass and were more ornamented than unmated females. They also found that the standardized variance in mating success as measured by microsatellite markers was at least seven times greater in females than in males. Despite morphological and genetic evidence supporting a female-biased PRR, it is not known whether male and female gulf pipeﬁsh do indeed differ in their respective potential reproductive rates. If the assumptions underlying sexual selection theory are correct, they should be supported both in species with conventional mating systems and in those that are sex-role reversed (Clutton-Brock 2009). The present study was designed to test the hypothesis that the sex differences in body size, ornamentation and parental roles of this species are accompanied by a sex difference in PRR. The 2-month study period allowed us to measure PRR over multiple breeding bouts. We predicted that females would have a higher PRR than males and that the rates in both sexes would change over successive breeding bouts in relationship to previous reproductive output. METHODS Animals We collected sexually mature S. scovelli adults (N ¼ 116) from sea grass beds in Sarasota Bay, Florida on 6 June 2004. Sexually mature males have a developed ventral brood pouch and females have complete silver bars on the trunk (Brown 1972; Jones & Avise 1997). Pipeﬁsh were collected with push nets (1 m2, with 2 mm2 mesh) at a depth of roughly 1 m. Fish were shipped overnight to the University of Oklahoma where they were housed in temporary, nonreproductive groups in aquaria that did not share a common water system. Most ﬁsh were allowed to acclimate to laboratory conditions for 10 days before the study began. Five focal females spent an additional 10–18 days acclimating while we waited for a sufﬁcient number of ﬁeld-pregnant males to return to breeding condition to pair with them. Animal Housing Fish were housed in 75-litre aquaria divided with opaque Plexiglas either in half or into a small (18.75-litre) and a large (56.25-litre) compartment (depending on treatment, see below). Aquaria contained both biological sponge ﬁlters and undergravel ﬁlters and were supplemented with saltwater bacteria once per week. Aquaria contained crushed coral and sand substrate, plastic plants that mimic natural sea grass (Thallassia sp.) and 15 cm long by 3 cm wide sections of grey PVC tubing cut in half for cover. Fish were fed newly hatched and adult Artemia enriched with Selco (Aquatic Ecosystems, Apopca, FL, U.S.A.) twice daily. The feeding regime was sufﬁcient to maintain a body mass to total length (TL) ratio that was similar to that found in the ﬁeld (S.K.S., unpublished data). Fish were kept at 25 C and on a 13:11 h light:dark cycle to mimic natural conditions in Sarasota Bay during the summer. Study Design We conducted a 61-day study where we provided focal male and female gulf pipeﬁsh with a surplus of mates and counted the Author's personal copy S.K. Scobell et al. / Animal Behaviour 78 (2009) 747–753 number of potential offspring each sex could produce. At the start of the study, 12 male and 12 female focal subjects were chosen to represent the range of body sizes for adult breeding individuals in the population (TL ranges: males 119–145 mm, N ¼ 68; females 112–169 mm, N ¼ 48). Focal animals’ TL (134.7 2.6 mm) did not differ from that of the population sample (132.4 1.2 mm; Mann– Whitney U test: U ¼ 1067.0, N1 ¼ 24, N2 ¼ 92, P ¼ 0.58). Focal animals were provided with similarly sized (within approximately 5 mm TL) opposite-sex partners because size-assortative mating has been documented in several species of syngnathids (Vincent & Sadler 1995; Jones et al. 2003; Silva et al. 2008). The number of partner males or females that each sex could mate with in 1 day was determined in a pilot study in the autumn of 2003. Females transferred eggs to the pouches of a maximum of two males per day; males only accepted eggs from one female on the ﬁrst day of the pregnancy. In the current study, to ensure that focal animals had a surplus of mates, each focal female was housed with three breeding males and each focal male was housed with two breeding females. Focal males were housed in larger compartments (56.8 litres) than focal females (37.5 litres) to reduce animal density and thus reduce female–female competition (between a focal male’s two partner females; S.K.S., personal observation) as this female competition was not the focus of the present study and could have interfered with assessment of PRR. On the four occasions when a focal male failed to get pregnant, one of his partner females was removed (the suspected subordinate) and replaced with a new partner female. This usually resulted in a new pregnancy the next day (N ¼ 3). Male–male aggression has not been observed in this species, so we were able to house the focal females and their partner males in smaller compartments than those used for the focal males and their partner females. As the study progressed and the number of available breeding partner males became limited (due to many males being pregnant), females were housed with at least two breeding males, the maximum number of males that a female could mate with in one day. All focal and partner males were checked daily between 1600 and 2000 hours for new pregnancies. Pregnancies were easily observed as the orange eggs are clearly visible though the skin of the male’s pouch. The day that the pregnancy was detected was designated day 0. Pregnant focal males remained in their home tank with both females. Partner males, after becoming pregnant, were moved to an adjacent 37.5-litre compartment, and a new partner male in breeding condition was placed in with that focal female. For all pregnant males (focal and partner), at mid-pregnancy we counted the eggs transferred by the female, and then, following birth, we counted the total number of offspring. To obtain the mid-pregnancy count, we counted the number of fertilized eggs and any unfertilized eggs on or around day 7 of the pregnancy; at this time, embryos are pigmented and fertilized eggs can be distinguished from unfertilized eggs. The males were lightly anaesthetized using a 0.05% solution of 2-phenoxyethanol (2-PE) and the contents of the pouch were counted through the transparent skin folds. On day 12 of pregnancy, pregnant males were placed in breeding nets containing a plastic plant and a PVC shelter in their home aquaria. Breeding nets and the male’s pouch were checked daily to determine whether the male had given birth. On the day of birth, the pouch contents (newborn, underdeveloped (dead) offspring and unfertilized eggs) were counted, newborn were euthanized with an overdose of 2-PE, and all pouch contents were then stored in 10% buffered formalin. After the 61-day study period, we recorded TL, snout–vent length (SVL), body depth just anterior to the dorsal ﬁn, and total body mass for the focal male and females. We euthanized focal males and females with an overdose of 2-PE and dissected the gonads, which we preserved in 10% formalin and later weighed to the nearest 0.1 mg. 749 Two focal females did not mate during the study, probably because of reproductive suppression (regression of the gonads in group-housed females; e.g. Rosenqvist 1990) during acclimation, and we removed them from analyses. This left 10 females in all analyses with the exception of analyses involving gonad mass. For these analyses, sample sizes were reduced (males: N ¼ 10, females: N ¼ 7) because of missing gonad data for several animals. The methods used in this study comply with the current laws of the United States of America, in which the studies were performed, and were approved by the University of Oklahoma Institutional Animal Care and Use Committee (R02-013). Analyses We calculated the PRR by measuring the potential offspring produced per unit time. The gulf pipeﬁsh has a courtship period that can be extremely short. Under laboratory conditions, the time from introduction of a partner to mating can be less than 1 min. Thus, individual mating events can be easily missed without 24 h observation, which was not logistically possible in this study. For each subject, we calculated total fecundity over the study period. To obtain the best measure of the focal animal’s fecundity for each pregnancy, we compared the mid-pregnancy counts to the pouch contents following birth, and then used whichever count was higher in our analyses. This approach minimized the error of the mid-pregnancy counts where some eggs, or occasionally an entire row of eggs, could be occluded from the scorer’s view. We calculated the PRR as each animal’s fecundity/day. However, as PRR is a measure of an individual’s potential offspring, we calculated the PRR for females as the number of eggs transferred/ day (for mid-pregnancy count) or all pouch contents (for count at birth), and for males, we calculated the PRR as the number of fertilized eggs/day (for mid-pregnancy count) or newborn þ underdeveloped young (for count at birth), whichever was greater. We used Student’s t test to determine whether there was a sex difference in mean PRR between males and females for the entire study period. For the repeated measures analysis, we divided the PRR data over the 61-day study into four breeding bouts. A breeding bout was deﬁned as the time that it took for males to complete a pregnancy (from mating to birth and thus return to breeding condition, N ¼ 12, mean SE ¼ 15.0 1 days). The PRR for each of the four breeding bouts was compared for males and females using repeated measures mixed model analysis (see Brown & Prescott 1999). Because most populations of S. scovelli show sexual size dimorphism (Reid 1954; Brown 1972; Jones et al. 2001), we used a principal components analysis (using PC-ORD, MJM Software Design, Gleneden Beach, OR, U.S.A.) to obtain a composite score for each focal animal’s body mass, depth, TL and SVL. Axis 1 explained 91.6% of the total variance in focal animals’ body size. We ﬁtted the body size PCA axis 1 scores and subjects (nested within sex) as random, so the effects of sex, breeding bout, PCA, and the interaction between sex*bout and sex*PCA on PRR could be assessed across focal animals. We used least squares means with post hoc Tukey–Kramer tests for sex and breeding bout. To assess the relationship between morphological measurements and PRR, we used linear regression analysis to determine whether TL, somatic mass (total mass gonad mass) or gonad mass affected the PRR for each sex. We used correlation analysis to determine the relationship between TL, somatic mass and gonad mass for each sex. The mixed model analysis was conducted using SAS 8.01 (SAS Institute, Inc., Cary, NC, U.S.A.). All other analyses were conducted using SPSS 15.0 (SPSS, Inc., Chicago, IL, U.S.A.). Author's personal copy 750 S.K. Scobell et al. / Animal Behaviour 78 (2009) 747–753 RESULTS Overall PRR Females mated on average 2.8 times more often than males over the course of our study (mean SE; females: 11.3 1.2, males: 4.1 0.2; Mann–Whitney U test: U ¼ 0.0, N1 ¼ 10, N2 ¼ 12, P < 0.001; Fig. 1a). Accordingly, females had a shorter mean interbrood interval (number of days between matings ¼ 6.3 0.8 days) than males (16.1 0.6 days, Student’s t test: t20 ¼ 10.09, P < 0.001; Fig. 1b). The males’ longer interbrood interval was due mainly to the length of pregnancy, rather than limited by female behaviour, 12 10 PRR and Body Size (a) Number of pregnancies PRR over Four Breeding Bouts The mean PRR of males and females did not remain constant over the four breeding bouts (Fig. 2). A repeated measures mixed model analysis showed that the interactions of sex*bout and sex*PCA were not signiﬁcant (sex*bout: F3,57 ¼ 0.52, P ¼ 0.67, sex*PCA: F2,17 ¼ 2.18, P ¼ 0.14); these terms were removed from the model and it was rerun. The main effects of sex and breeding bout both affected PRR (sex: F1,20 ¼ 21.79, P ¼ 0.0001; bout: F3,63 ¼ 3.10, P ¼ 0.03). Post hoc analyses showed that the PRR of breeding bout 1 was signiﬁcantly greater than the PRR for breeding bout 2 (least squares means with Tukey–Kramer adjustment: t63 ¼ 2.92, P ¼ 0.005). The decrease in PRR from the ﬁrst to second breeding bout tended to be greater for females (4.0 1.4) than males (1.9 0.7) (Student’s t test: t20 ¼ 4.28, P ¼ 0.052). The PRR for breeding bout 2 was also signiﬁcantly lower than the PRR for breeding bout 3 (t63 ¼ 2.21, P ¼ 0.03). However, the change in PRR between the second and third breeding bouts did not differ signiﬁcantly between females (3.2 1.0) and males (1.3 0.6) (Student’s t test: t20 ¼ 0.70, P ¼ 0.41). For both male and female focal animals, we noticed a common pattern of fecundity over the four breeding bouts. Most individuals had high fecundity during the ﬁrst and third breeding bouts, but much lower fecundity in the second breeding bout (see Fig. 3). Not all individuals showed this pattern, but all showed at least one ‘boom’ and then ‘bust’ in fecundity over consecutive breeding bouts during the study. 14 * 12 10 8 6 4 2 0 18 Interbrood interval (days) because focal males were pregnant within 2.0 0.7 days of giving birth (median ¼ 0.0 days; most males got pregnant the day after giving birth). Females had a mean PRR 1.95 times greater than that of males over the entire study period of 61 days. Focal females had a mean PRR of 6.5 1.1 offspring/day, whereas focal males had a mean PRR of 3.4 0.3 offspring/day (Student’s t test: t20 ¼ 2.99, P ¼ 0.01; Fig. 1c). 16 (b) * 14 8 Although the ranges of male and female body size measurements overlapped for the focal animals in this study, females in our sample, as in other studied populations (Reid 1954; Joseph 1957; 6 4 2 12 0 PRR (offspring/day) (c) Mean PRR (offspring/day) 8 * 6 4 2 P = 0.005 10 8 6 4 2 1.90:1 0 0 Females Males Figure 1. Mean SE (a) number of pregnancies, (b) interbrood interval and (c) potential reproductive rate (PRR) of female and male gulf pipeﬁsh over the 61-day study. N ¼ 10 females, N ¼ 12 males. *P < 0.01. P = 0.03 1 1.77:1 2.05:1 2 3 Breeding bout 2.03:1 4 5 Figure 2. Potential reproductive rates (PRR) for female (C) and male (B) gulf pipeﬁsh over four breeding bouts (mean SE). The ratio of female to male PRR is shown for each breeding bout. Brackets and associated P values denote differences in the PRR between the indicated time periods. Author's personal copy 140 16 120 14 PRR (offspring/day) Fecundity (offspring/pregnancy) S.K. Scobell et al. / Animal Behaviour 78 (2009) 747–753 100 80 60 40 12 10 8 6 4 2 20 0 751 21 Jun 2004 5 Jul 2004 0 50 19 Jul 2004 2 Aug 2004 Figure 3. Representative fecundity data from an individual female and an individual male gulf pipeﬁsh. Each data point represents a single pregnancy over the course of the study. Female 19 (C) and male 11 (B) best exempliﬁed the ‘boom’ and ‘bust’ mating patterns observed in the focal animals as well as the sex differences in mating frequency. Brown 1972), were generally larger than males (Table 1). However, neither female TL (linear regression: R2 ¼ 0.01, P ¼ 0.81) nor somatic mass (R2 < 0.01, P ¼ 0.91) was related to the PRR. Ovarian mass also did not correlate with female TL (Spearman rank correlation: rS ¼ 0.07, N ¼ 7, P ¼ 0.88) or somatic mass (rS ¼ 0.11, N ¼ 7, P ¼ 0.82), but females with a greater ovarian mass also had a higher PRR (R2 ¼ 0.77, P ¼ 0.01) (Fig. 4). For males, testes mass increased with both TL (Spearman rank correlation: rS ¼ 0.94, N ¼ 10, P < 0.01) and somatic mass (rS ¼ 0.95, N ¼ 10, P < 0.01). However, larger males and males with larger testes did not have a greater PRR (TL: R2 < 0.01, P ¼ 0.82; somatic mass: R2 < 0.01, P ¼ 0.99; testes mass: R2 < 0.01, P ¼ 0.96). DISCUSSION Our study documented that the PRRs of male and female gulf pipeﬁsh differ, with females having an overall PRR almost twice that of males. As in other sex-role reversed species of syngnathids (Berglund et al. 1989; Kvarnemo & Ahnesjö 1996), female gulf pipeﬁsh are able to mate more often and with a shorter interbrood interval than are males. The gulf pipeﬁsh mating system is consistent with sexual selection theory in that the sex that has the higher PRR, females, also has the higher variance in mating success (Jones et al. 2001) and is the more ornamented sex (Reid 1954; Brown 1972; Jones et al. 2001; Clutton-Brock 2009). Assuming a 1:1 adult sex ratio and no differential mortality, we predict that gulf pipeﬁsh have a female-biased OSR in the ﬁeld (Jones & Avise 1997; Ahnesjö et al. 2001; Jones et al. 2001) and, accordingly, that females Table 1 Comparison of morphological measurements of focal male and female gulf pipeﬁsh following the study period of 61 days (mean SE) Females 10 59.82.0 140.24.8 7.20.3 2.30.2 116.930.9 Males 12 49.41.0 127.62.5 5.50.2 1.80.1 5.40.5 *P < 0.05; **P < 0.01. y Sample size for gonad mass: females N ¼ 7, males N ¼ 10. 150 200 250 300 Ovary mass (mg) Date of mating Sample size (N) Snout–vent length (mm) Total length (mm) Depth of trunk (mm) Body mass (g) Gonad mass (mg)y 100 t P 5.01 2.44 4.45 1.98 4.37 <0.01** 0.02* <0.01** 0.06 0.01* Figure 4. Potential reproductive rate (PRR) versus ovarian mass for focal gulf pipeﬁsh females. court males and compete with each other for access to mates (Jones & Avise 1997; Clutton-Brock 2009), as we have observed in the laboratory. Although it has been suggested previously (Ahnesjö et al. 2001), the present study is, to our knowledge, one of the few studies to measure PRR over several breeding bouts in a vertebrate. The PRR of gulf pipeﬁsh males, and particularly females, varied over the four breeding bouts. The mean PRR was highest for both sexes during the ﬁrst breeding bout. Then, both sexes showed a decrease in mean PRR during the second breeding bout. This general pattern of a boom followed by a bust in reproductive investment was seen for most of our focal animals. The change in male and female PRR over the study is consistent with life-history theory predictions that individuals that mate multiply make trade-offs between current and future reproductive investment given a limited lifetime energy budget and potential availability of mates (Stearns 1992; Zera & Harshman 2001; Roff 2002; Heubel et al. 2008). However, it is also possible that this pattern of reproduction was a result of the laboratory environment. Future studies of this species in the ﬁeld and in the laboratory will help clarify the factors that contribute to the variation in PRR that we observed across multiple bouts of mating. Saltwater populations of gulf pipeﬁsh generally show sexual size dimorphism (Reid 1954; Joseph 1957; Brown 1972). Our sample from the Sarasota Bay population also showed sexual size dimorphism, with females, on average, larger than males. Larger female body size is common in ﬁsh and is often related to a fecundity advantage (Andersson 1994). When each sex was examined individually, neither male or female total length nor somatic mass affected the PRR. However, males and females did differ with respect to the relationship of gonad size to PRR. Females that had larger ovaries had a higher PRR, whereas males with larger testes showed no concomitantly greater PRR. A female with larger ovaries probably has a greater ﬁtness advantage over a female with smaller ovaries. Females with larger ovaries can transfer more eggs, and because males in this species typically only accept eggs from one female (Jones & Avise 1997; Jones et al. 2001), they would be predicted to choose the female that can transfer the greater number of eggs (Rosenqvist 1990). As a result of male pregnancy, males with bigger testes probably do not gain a signiﬁcantly greater ﬁtness advantage over males with smaller testes. Males in this species are thought to fertilize the eggs once they are in the male’s pouch, similar to fertilization in S. schlegeli (Watanabe et al. 2000). There has been no record of multiple paternity in S. scovelli (Jones & Avise 1997; Jones et al. 2001). Thus, there appears to be no sperm Author's personal copy 752 S.K. Scobell et al. / Animal Behaviour 78 (2009) 747–753 competition within the brood pouch and sperm probably do not have to fertilize eggs in the external environment. This speculation is also supported by the very small size of testes in this species. Our results are consistent with sexual selection acting more strongly on female ovary size, with relaxed selection on male testis size (Clutton-Brock 2009). The difference in PRR between the sexes in gulf pipeﬁsh is presumably inﬂuenced by the differing constraints of their reproductive physiology. Males are constrained by the time that it takes to brood offspring, whereas females are only constrained by the time that it takes to produce another batch of mature eggs. In our study, a male’s ‘time out’ was consistently 14–15 days for each pregnancy. Females, in contrast, were able to remate rapidly. For example, one female mated with eight males within a 2-week period, delivering full clutches of eggs (enough to ﬁll the male’s brood pouch) to ﬁve of them. To be able to mate multiply in rather rapid succession, females must have a reproductive system that facilitates successive rounds of ovulation in a short time period. The ovary of the gulf pipeﬁsh is unusual because it is structured in a conveyor belt fashion (Begovac & Wallace 1987). As one set of eggs is ovulated into the lumen, the next set moves into its place and completes the ﬁnal stage of maturation (Begovac & Wallace 1988). The anatomy suggests that female gulf pipeﬁsh may have a very short period of time between the ovulation of each set of oocytes. However, we are unaware of any published studies on the length of the female reproductive cycle or the hormonal mediation of maturation of oocytes and ovulation in this species. Recent research does suggest a close link between ovarian morphology and PRR within the family Syngnathidae. Sea horses and the monogamous pipeﬁsh C. haematopterus have ovaries that differ in morphology from those of the gulf pipeﬁsh. Recently, Sogabe et al. (2008) found that C. haematopterus females have ovaries with two germinal ridges, similar to ovaries in sea horses (Selman et al. 1991), but not to ovaries of other pipeﬁsh in the genus Syngnathus. These authors suggested that this structural difference might relate to the functional difference in reproduction between polyandrous/polygynandrous Syngathus pipeﬁshes with female-biased PRRs versus the monogamous sea horses and C. haematopterus pipeﬁsh with PRRs that approach unity. The group-synchronous ovaries of the monogamous sea horses and C. haematopterus result in a longer ‘time out’ for these females (Hippocampus spp.: 9–45 days, Foster & Vincent 2004; C. haematopterus: 10–19 days, Sogabe et al. 2007) than for Syngnathus females (48 h, Berglund et al. 1988; present study) that have an asynchronous-type ovary (Wallace & Selman 1981; Begovac & Wallace 1988). It is possible that these two types of ovarian morphologies dictate the speed at which females of these species can produce mature eggs, which would directly affect the PRR. Further studies are needed to determine how the reproductive physiology of the ovarian cycle relates to these two distinct ovarian morphologies and whether ovarian morphology and/or physiology produce functional constraints that affect the PRR in these species. Acknowledgments We thank P.L. Schwagmeyer and Edith Marsh-Matthews for their help with the analysis and comments on the manuscript. 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