Interspawning interval of wild female three-spined stickleback Gasterosteus aculeatus in Alaska

Journal of Fish Biology (2009) 74, 2299 2312 doi:10.1111/j.1095-8649.2009.02237.x, available online at www.interscience.wiley.com Interspawning interval of wild female three-spined stickleback Gasterosteus aculeatus in Alaska N. J. Brown-Peterson*, and D. C. Heins Department of Coastal Science, The University of Southern Mississippi, 703 East Beach Dr., Ocean Springs, MS 39564, U.S.A. and Department of Ecology and Evolutionary Biology, 400 Lindy Boggs Center, Tulane University, New Orleans, LA 70118, U.S.A. (Received 28 March 2008, Accepted 16 February 2009) The interspawning interval, or spawning frequency, of wild three-spined stickleback, Gasterosteus aculeatus, was estimated using histological examination of postovulatory follicles (POF). Females in Alaskan lakes appeared to have as much as a 48 h delay between ovulation and ovoposition, yet the POF method could still be used to estimate the interspawning interval. In two Alaskan lakes the interspawning interval was estimated to range from 2 2 to7 8 days among individual female G. aculeatus. These estimates were consistent with the range (2 5 to 5 days) of previous estimates among individual females from laboratory observations of spawning G. aculeatus, aswell as anecdotal accounts of spawning intervals reported from wild populations in Canada (5 10 days). The interspawning interval of females increased during the course of the spawning season in Alaska, showing that the majority of female spawning activity occurred during the earliest portion of the approximate 6-week reproductive season. The increased interspawning interval appears to be related to a previously reported decrease in body condition in reproductive females during the breeding season. Thus, female G. aculeatus may be unable to sustain the initial rate of reproduction as energy stores that support the rapid growth of vitellogenic oocytes are depleted. Journal compilation 2009 The Fisheries Society of the British Isles Key words: clutch frequency; interbrood interval; reproductive behaviour; reproduction; spawning frequency. INTRODUCTION The three-spined stickleback Gasterosteus aculeatus L. has been studied extensively as a model for evolutionary studies because of its widespread geographic distribution and the variability of freshwater populations in morphology, physiology and life history characteristics throughout the range (Foster & Baker, 2004; Gibson, 2005). Reproduction of G. aculeatus has been comprehensively reviewed (Wootton, 1976; Baker, 1994; Baker et al., 2008), and the production of multiple clutches of eggs by G. aculeatus during the spawning season is well established (Wootton, 1976). Nontheless, aspects of the reproductive ecology of the species remain relatively unknown. For instance, the interspawning interval, or interbrood interval, of females has not been quantified for any wild populations. The interspawning interval is an *Author to whom correspondence should be addressed. Tel.: +1 2288724288; fax: +1 2288724204; email: nancy.brown-peterson@usm.edu 2299 Journal compilation 2009 The Fisheries Society of the British Isles

2300 N. J. BROWN-PETERSON AND D. C. HEINS important life history trait because the total annual fecundity of a female, which in many cases may represent life-time fecundity for G. aculeatus, is determined by the size of the clutches and the number of clutches produced in a given spawning season. The determination of the interspawning interval, also known as spawning frequency, is routine in marine fishes that spawn pelagic eggs in multiple batches. The classic method for determining spawning frequency involves histological evaluation of postovulatory follicles (POF) in ovaries of fishes during the reproductive season (Hunter & Macewicz, 1985). This methodology has not been applied to freshwater fishes, although histological analysis of POF has been used to provide a more accurate assessment of the timing and duration of the spawning season in a variety of stream fishes (McAdam et al., 1999; Brewer et al., 2006, 2008). Generally, estimates of interspawning interval for small freshwater species are limited to direct observations of individual fishes in the laboratory (Wootton, 1973, 1974a; Heins & Rabito 1986; Fletcher & Wootton, 1995; Ali & Wootton, 1999). Estimates of interspawning interval in wild freshwater fishes have been limited to observations on multiple recaptures of marked fishes (Bolduc & FitzGerald, 1989; Fletcher & Wootton, 1995) and experiments using in-stream cages (Weddle & Burr, 1991). Doubtless, adoption of a direct estimate of interspawning interval would greatly improve estimates of this important life history trait in freshwater species. Studies addressing the reproductive biology of G. aculeatus in the Matanuska- Susitna (Mat-Su) Valley of south-central Alaska have provided information about the reproductive characteristics of G. aculeatus in the region (Baker et al., 1998; Baker & Foster, 2002; Baker et al., 2005, 2008). Although these fish in Alaska are known to have a short spawning season of c. 6 weeks (Heins et al., 1999), the interspawning interval of females in these natural populations remains unknown. Furthermore, techniques for estimating the interspawning interval in wild populations of G. aculeatus are lacking. The first objective of the current study was to ask whether the histological method of estimating interspawning interval using POF, as commonly employed for marine fishes, can be applied to G. aculeatus. Answering this question involves comparing estimates of interspawning interval obtained for wild populations using histology to known interbrood intervals from laboratory studies. The second objective was to ask whether estimates of interspawning interval vary over the course of the spawning season within the same population of fish. During the course of the reproductive season, female body condition in reproductive G. aculeatus decreases as energy reserves presumably are depleted (Bagamian et al., 2004). Thus, the hypothesis that interspawning interval increases during the spawning season was tested. STUDY AREA MATERIALS AND METHODS Samples were obtained from, and field experiments were conducted in Wolf Lake (61 38 39 N; 149 16 20 W) and Walby Lake (61 37 14 N; 149 12 48 W), which are located in the Mat-Su Valley north of the Cook Inlet, Alaska. Wolf Lake has a surface area of 25 ha and Walby Lake has a surface area of 22 ha. The lakes lie among many lakes and ponds that dot the glacial moraine of the Mat-Su Valley. Lakes in the region are usually covered with ice from approximately October into May (Woods, 1985). The average July air

INTERSPAWNING INTERVAL OF ALASKAN G. ACULEATUS 2301 temperature from two distant stations in the Mat-Su Valley is c. 14 15 C (Woods, 1985), and the average warmest summer temperatures throughout the area are c. 18 20 C(Reger & Updike, 1983). FISH COLLECTIONS Samples of G. aculeatus were obtained from Walby and Wolf Lakes between late May and early June of 2000 and 2001. This time of year approximates the firsthalfofa6 week spawning season that occurs after ice-out in Alaska (Heins et al., 1999). Collections were made near shore by setting 6 mm un-baited wire-mesh minnow traps overnight. Fish were anaesthetized until quiescent in MS-222. Each specimen was prepared for histological examination by making an oblique cut through the caudal peduncle and cutting open the abdomen before placing the fish in a histological mega-cassette for fixation. The fish were fixed and stored in 10% phosphate-buffered formalin until laboratory examination. To determine the time of day G. aculeatus ovulate and to estimate ages of POF, time-course experiments were conducted in Walby Lake from 31 May to 1 June 2000 and in Wolf Lake from 4 to 5 June 2000. Minnow traps were pulled to remove adult females for histological samples, emptied of other fish, and re-set at c. 6 h intervals for a total of four samples during a 24 h period. Another time-course experiment was conducted in Wolf Lake from 4 to 8 June 2001 to verify the ageing of POF and the spawning periodicity estimates. Gravid G. aculeatus were captured in minnow traps, gently stripped of their eggs, and returned to the traps. Ten females were anaesthetized in MS-222 and fixed in a formalin solution soon after their eggs were stripped (time 0 h). Approximately 10 females were then removed from traps every 12 h for the next 72 h to be anaesthetized and fixed (times 12, 24, 36, 48, 60 and 72 h). Data collected from females infected with the cestode macroparasite Schistocephalus solidus Müller were removed prior to analysis. HISTOLOGICAL METHODS Ovaries were removed from each fish in the laboratory and staged macroscopically following Baker et al. (1998) and Heins et al. (1999). Whole ovaries were placed into individually labelled cassettes, rinsed overnight in running tap water, dehydrated in a series of graded ethanols and embedded in paraffin following standard histological procedures. Ovarian tissue was sectioned at 4 μm and stained with haematoxylin 2 and eosin Y (Richard Allen Scientific; www.rallansci.com) for histological inspection. Histological ovarian phases were assigned following Brown-Peterson et al. (2007). These phases correspond to the traditional G. aculeatus ovarian-oocyte classification (Heins et al., 1999) as follows: the developing phase contained early and late maturing oocytes (EM and LM ovaries, respectively), the spawningcapable phase contained mature and ripening oocytes (MA and MR ovaries, respectively), and the actively spawning phase contained ripe eggs ovulated from their follicles but still contained within the ovary (RE ovaries). Oocytes were considered to be undergoing oocyte maturation (OM) when nuclear migration was evident and the yolk granules had fused and become liquid yolk; for G. aculeatus, this occurs at the end of the spawning-capable phase and is similar to the MR ovarian stage definedbybakeret al. (1998) and Heins et al. (1999). Ages were assigned to POF based on fish examined from the time-course experiments as well as original descriptions of POF in Hunter & Macewicz (1985) for the northern anchovy Engraulis mordax Girard. WATER TEMPERATURES RESULTS Water temperatures recorded during the study were similar in Walby and Wolf Lakes. In 2000, temperatures ranged from 13 to 15 C in Wolf Lake and from 12 to 16 C in Walby Lake at the times of fish collections. In 2001, temperatures ranged

2302 N. J. BROWN-PETERSON AND D. C. HEINS (a) (b) (c) (d) (e) (f) FIG. 1. Histological sections showing postovulatory follicles (POF) of wild-caught Alaskan Gasterosteus aculeatus. Fish were stripped of ovulated eggs, placed in cages and sampled every 12 h to verify POF age: (a) 6 12 h POF, (b) 24 h POF, (c) 36 h POF, (d) 48 h POF, (e) 3 day POF and (f) 4 day POF. Haematoxylin and eosin staining; arrows indicate POF. from 14 to 17 C in Walby Lake during 29 30 May. Temperatures increased slightly in Wolf Lake during the collection period, ranging from 14 to 16 C during 29 30 May and 15 to 17 5 C from 4 to 8 June. Ice was completely melted from both lakes during the study; exact dates of ice-out for these lakes are unavailable. POF AGES Gasterosteus aculeatus POF look similar to previous descriptions of POF from E. mordax. Due to the cold water temperatures in Alaskan lakes, however, POF degradation is much slower than in more temperate climates, which allows accurate determination of POF up to 4 days in age. Fig. 1 shows typical G. aculeatus POF stages, ranging in age from 6 to 12 h up to 4 days. The ages of these POF were verified by stripping eggs from 60 gravid females, returning them to cages and sampling the fish throughout a 72 h time-course experiment. At the time of initial stripping, ovaries of 70% of the fish in the first subsample (time 0 h) contained some

INTERSPAWNING INTERVAL OF ALASKAN G. ACULEATUS 2303 TABLE I. Postovulatory follicle (POF) ages determined from caged, stripped Gasterosteus aculeatus. Data (ovulated or POF) are expressed as percentages of total number of fish stripped n at each time point Time since 6 12 h 24 h 36 h 48 h 3 days 4 days stripping (h) n Ovulated POF POF POF POF POF POF 0 10 70 30 20 30 20 12 10 20 50 20 20 10 24 10 30 50 50 36 9 11 22 67 48 10 20 50 30 20 60 9 11 22 44 34 72 9 11 11 89 ovulated eggs as well as POF of multiple ages (Table I). Only 30% of these females had new 6 to 12 h POF, suggesting ovulation had occurred within the past 24 36 h for the majority of the fish. These data indicate that G. aculeatus are capable of holding ovulated eggs in the ovary for up to 2 days prior to oviposition. Thus, fish with ovulated eggs in the ovary can reasonably be expected to have POF from those eggs ranging in age from 6 to 12 h up to 36 h. Females sampled 12 h after egg stripping did not show any POF classified as 6 to 12 h, although 50% of the fish had 24 h POF (Table I). This suggests the 6 to 12 h POF stage is transitory in nature and that POF may enter the 24 h POF stage sooner than 24 h post-ovulation. The occurrence of 20% of the fish in this sample with some ovulated eggs suggests the time 0 h stripping of eggs was not as complete as anticipated, and can also account for the presence of 24 or 36 h POF in the 12 h post-stripping samples. Additionally, some of the old POF (i.e. 3 and 4 days) in the ovaries of the 12 h samples no doubt represent ovulation of a previous clutch prior to the fish being stripped. All females examined 24 h after egg removal had POF classified as 24 or 36 h (Table I), indicating these POF classifications accurately represent time since ovulation. Similarly, the majority of fish examined 36 h post-egg removal had POF classified as 36 h (Table I). Females sampled 48 h post-egg removal had POFs in three age classes, ranging from 36 to 72 h (Table I). Twenty per cent of these fish had ovulated eggs in the ovary; this could account for the higher percentage of 36 h POF than expected. At 60 h post-stripping, only 34% of the fish had POF classified as 60 72 h, an unexpected result. By 72 h post-stripping, however, 89% of the fish examined had POF in the 60 72 h class (Table I), verifying that POF up to 3 days old can be accurately aged in G. aculeatus. OVULATION TIME Examination of 146 G. aculeatus from Wolf and Walby Lakes in 2000 with ovaries containing ovulated eggs and POF showed that females can ovulate their eggs up to 48 h prior to releasing them from the ovary for ovoposition. Fish captured over a 24 h period had POF with a variety of ages in their ovaries (Fig. 2), although the highest percentages of POF at the 1000, 1600 and 2100 hour time points corresponded to a 1000 hour ovulation time. When the POF ages displayed in Fig. 1 are backcalculated

2304 N. J. BROWN-PETERSON AND D. C. HEINS 60 Per cent of fish in POF stage 50 40 30 20 10 0 1000 1600 2100 0400 Time of day (hours) FIG. 2. Ovulation time of wild-caught Alaskan Gasterosteus aculeatus as determined from postovulatory follicle (POF) ages 6 12 h POF; 24 h POF; 36 h POF; 48 h POF. Data are from fish uninfected by Schistocephalus solidus parasites with ovulated eggs in the ovary. All fish were captured in Wolf and Walby Lakes in 2000. to a time of day (Table II), most fish appeared to have ovulated within a 6 h period during the morning (0400 1000 hours). SPAWNING PERIODICITY Spawning periodicity was inferred from POF age in gravid but non-ovulated females. Fish with POF 36 h were considered to have spawned <2 days previously, a 2 day interspawning interval corresponded to fish with 48 h POF, a 3 day interval corresponded to fish with 60 72 h POF, a 4 day interval corresponded to fish with 4 day old POF, and fish classified as spawning >4 days previously had no POFs in the ovary. Estimates from 2000 data clearly show the highest percentage of fish with a 4 day spawning periodicity (Fig. 3). Interspawning interval estimates were more variable for fish captured in 2001, although the highest percentage had a spawning interval of >4 days. A large percentage of fish captured in 2001, however, appeared to be spawning-capable every 2 days, indicating a wide variation in spawning periodicity among females in the population. Another, and perhaps more standard, method of estimating spawning interval is to look at the percentage of females in spawning condition with ovaries containing either oocytes undergoing oocyte maturation (OM) or with POF that are <24 h, representing females in which ovulation is imminent (OM) or those that have just completed ovulation (POF < 24 h). All females for this analysis had ovulated eggs in the ovary. No fish were found undergoing OM in Wolf Lake in 2000, but estimates using POF <24 h suggest a 4 3 day spawning interval (Table III). This estimate is similar to estimates obtained from gravid, non-ovulated females in the same collection (Fig. 3). Females collected at the beginning of the spawning season (29 30 May) in 2001 from both Wolf and Walby Lakes yielded estimates of spawning interval ranging from 2 2 to4 5days (Table III), which are similar to previous estimates using the POF method. Thus, two different methods show that some individual G. aculeatus in two Alaskan lakes are capable of spawning as frequently as every 2 days during

INTERSPAWNING INTERVAL OF ALASKAN G. ACULEATUS 2305 TABLE II. Estimated ovulation time in Alaskan Gasterosteus aculeatus based on percentage of fish containing postovulatory follicle (POF) during a 24 h study, (n = 146 fish) Time of day (hour) POF in ovary at time point (%) 0400 20 5 1000 53 5 1600 12 4 2100 13 7 the reproductive season, although a more reasonable estimate of the population mean would be a spawning periodicity of c. 4days. CHANGES IN SPAWNING INTERVAL DURING THE REPRODUCTIVE SEASON No G. aculeatus in the developing phase were found in the 7 8 June collection from Wolf Lake in 2001, and there was a greater percentage of females with regressing ovaries during this time than just 1 5 weeks earlier in the season (29 30 May; Table IV). Furthermore, there was a higher percentage of atretic oocytes in the ovaries of females in all reproductive phases from the later collection time, indicating the end of the spawning season. Although a high percentage of females from 7 8 June was in the actively spawning phase, a much lower percentage was undergoing OM than earlier in the season (Table IV), indicating fewer fish have oocytes undergoing the final phases of maturation later in the season. The interspawning interval of G. aculeatus appears to increase as the reproductive season progresses, based on data from Wolf Lake. Spawning frequency during the early portion of the reproductive season (29 30 May 2001) was estimated to be once every 2 to 3 days, which is more frequent than the estimate for 4 5 June 2000 from 60 Per cent occurrence POF stage 50 40 30 20 10 0 <2 2 3 4 >4 Age of POF (days) FIG. 3. Spawning interval estimates [based on per cent occurrence of postovulatory follicles (POF) stages] of wild-caught Alaskan Gasterosteus aculeatus in 2000 (, n = 35) and 2001 (, n = 48). All fish were capable of spawning but not ovulated and uninfected by Schistocephalus solidus parasites. Data from Wolf and Walby Lakes combined.

2306 N. J. BROWN-PETERSON AND D. C. HEINS TABLE III. Spawning interval estimates for Gasterosteus aculeatus from two Alaskan lakes based on two separate methods. Only fish uninfected by Schistocephalus solidus parasites were considered for this analysis Lake Date n OM (days) <24 h POF (days) Wolf 4 5 June 2000 129 4 3 Walby 29 30 May 2001 41 4 5 3 7 Wolf 29 30 May 2001 79 3 2 2 2 Wolf 7 8 June 2001 78 7 8 3 2 n, number of fish analysed; OM, oocytes undergoing final maturation just prior to ovulation; POF, postovulatory follicle. the same lake (Table III). Additionally, spawning interval estimates were greater for the Wolf Lake samples taken 7 8 June 2001. The OM method estimates an increase in spawning interval from 3 2 days in May to 7 8 days in June, while the POF method estimates an increase from 2 2 days in May to 3 2 days in June (Table III). A similar comparison could not be made for the 2001 Walby Lake collections because most fish collected in June were infected with S. solidus. DISCUSSION USE OF POSTOVULATORY FOLLICLES TO ESTIMATE INTERSPAWNING INTERVALS Data presented here demonstrate that histological estimations of spawning frequency can be applied to a freshwater species living in a cold water environment. This technique, previously used only in marine fishes, has wide applicability to a variety of freshwater species. The definition of the various ages of POF is an important component of effectively using POF to estimate spawning frequency. Hunter & Macewicz (1985) were the first to definitively characterize ages of POF, using G. mordax spawned in the laboratory. The technique presented here of calibrating POF from field-collected samples is effective and could be adopted for use in other species with reproductive strategies similar to G. aculeatus. Furthermore,the data demonstrate that POF can be used to estimate interspawning intervals in fishes that spawn large, demersal eggs rather than small, pelagic eggs as has traditionally been done (Hunter & Macewicz, 1985; Brown-Peterson, 2003; Waggy, 2004). TABLE IV. Reproductive phases of Gasterosteus aculeatus captured in Wolf Lake in 2001. Data are expressed as percentage of fish in each class Phase 29 30 May 2001 (n = 78) 7 8 June 2001 (n = 75) Developing 1 3 0 Capable of spawning (all) 48 7 40 0 Capable of spawning w/om 23 1 10 7 Actively spawning 48 7 54 6 Regressing 1 3 5 3 n, number of fish analysed; OM, oocyte maturation.

INTERSPAWNING INTERVAL OF ALASKAN G. ACULEATUS 2307 Spawning frequency has been successfully determined in marine fishes using both the POF and the OM methods (Hunter et al., 1986; Brown-Peterson et al., 2001; Brown-Peterson, 2003). Often, spawning frequency estimates are virtually identical using these two methods, but occasionally there are differences (Brown-Peterson et al., 2001, 2002) between estimates. Moreover, estimates do not consistently produce a longer or shorter spawning frequency estimate based on the POF method when compared to the OM method, suggesting the best method for estimating spawning frequency may be species-specific. The observed variations in estimates in different studies are most likely due to availability or catchability of fishes containing either POF or undergoing OM (Brown-Peterson et al., 2001). Thus, the differences in spawning frequency estimates between methods observed for G. aculeatus are not unexpected. The greater consistency among lakes and years when using the POF method suggests this may be the more consistent method for determining spawning frequency in G. aculeatus, as it is for spotted seatrout Cynoscion nebulous (Cuvier) (Brown-Peterson et al., 2002). The results of the POF method are considered to be the most reliable in this study. In this study, spawning interval was estimated to range between every 2 2 and 7 8 days for individual G. aculeatus in two Alaskan lakes. These estimates fall within the range (2 5 5 days) previously reported for individual females from laboratory observations of spawning fish (Wootton, 1974a, 1976), further validating the use of the POF method for estimating spawning frequency. Additionally, these estimates are similar to anecdotal accounts of spawning frequency reported from wild populations in Canada (5 10 days; Bolduc & FitzGerald, 1989). Food ration has a strong effect on spawning frequency of laboratory-reared G. aculeatus (Wootton, 1973, 1977; Fletcher & Wootton, 1995; Ali & Wootton, 1999), which accounts for the wide range of reported spawning intervals (3 9 days) among females in ration studies. TIMING OF OVULATION AND OVOPOSTION Gasterosteus aculeatus in Alaskan lakes appear to have up to a 48 h delay between ovulation and ovoposition, based on the age of POF and the presence of ovulated eggs in the ovary. This observation stands in contrast to an earlier supposition by Baker et al. (1998), who estimated the time between ovulation and ovoposition was more rapid (<12 h). The length of the observed period between ovoposition and ovulation means that spawning interval estimates based on POF will be an estimate of ovulation frequency rather than frequency of ovoposition. For the purposes of estimating spawning intervals in G. aculeatus, ovulation frequency is assumed to be closely related to spawning frequency even though they do not occur simultaneously as in marine fishes. Nonetheless, comparisons between these calculated spawning intervals and previously used interspawn intervals (Wootton, 1974a, 1976; Ali & Wootton, 1999) may be affected by the length of time between ovulation and ovoposition. Females that routinely deposit eggs within 12 h after ovulation will have a shorter interspawning interval than those that hold eggs for up to 2 days post-ovulation, assuming that females do not begin producing the next clutch until after oviposition instead of after ovulation. If females begin to yolk another clutch of eggs while carrying an ovulated but not oviposited clutch, the ovulation frequency should provide a good estimate of the spawning frequency. Indeed, the later situation appears to be the case with G. aculeatus, as Wallace & Selman (1979) suggested

2308 N. J. BROWN-PETERSON AND D. C. HEINS that the surge of gonadotropin, which induces oocyte maturation, also recruits a new clutch of vitellogenic oocytes. The ability of G. aculeatus to hold their eggs up to 48 h after ovulating the clutch before ovopositing them has important implications for reproductive behaviour in G. aculeatus. Ripe females, which carry ovulated eggs in the lumens of their ovaries and are ready to spawn, will swim among territories held by reproductive males as they sequentially assess individual males prior to mate selection (Wootton, 1974b, 1976; Foster 1994, 1995). During the process of finding and assessing males, an individual female s mate choice may be influenced by her energy reserves and available time (Bakker & Milinski, 1991; Milinski & Bakker, 1992; Luttbeg et al., 2001). The difference between time of ovulation and ovoposition thus allows a wider range of options in behavioral interactions between males and females. If spawning does not have to follow soon after ovulation, females will have more time to be choosy and males will have to be more overt in their means of female attraction. Moreover, as time passes after ovulation, females may become increasingly less choosy and more likely to spawn. Thus, variation in G. aculeatus reproductive behaviour would be expected, which is influenced by the time that has elapsed after ovulation, among individual females and is caused by the shifting selective forces on both males and females. VARIATION IN SPAWNING INTERVAL The POF estimates of spawning frequency reported here suggest that an individual female may spawn between five and 14 times during the c. 6-week reproductive period in Alaskan lakes, representing a substantial amount of energy allocated to reproduction. The number of spawns during a season can be influenced by female size, food availability, competition and temperature (Wootton, 1976). Moreover, interspawning intervals may also reflect consistent variation among females with some fish showing shorter or longer interspawning periods on average than others. For instance, laboratory observations of four spawning pairs of bannerfin shiner Notropis leedsi Fowler showed that spawning intervals averaged 3 3 4 1 days among individual females and varied as little as 3 4 days and as much as 2 6 days within individuals (Heins & Rabito, 1986). Additionally, the interspawning interval of 34 pairs (193 intervals) ranged from 3 to 10 days in another experiment with the same species (Heins & Rabito, 1986). Wootton (1974a) reported an interspawning interval averaging 3 6 days (range: 2 6 4 7 days) in 13 individual G. aculeatus in one laboratory experiment. Thus, overall variation in mean estimates of spawning frequency calculated for Alaskan G. aculeatus apparently reflects among and withinfemale variation in the samples of fish examined. Estimates of spawning frequency apparently also reflect seasonal variation arising within females. Gasterosteus aculeatus have a short reproductive season in Alaskan lakes (Heins et al., 1999), and the shorter spawning intervals calculated for the earliest portion of the season suggest the majority of reproductive activity in females occurs during this time. A difference in spawning intervals over the course of the reproductive season has not been well documented in any species, although data from estuarine spawning sciaenids such as C. nebulosus and silver perch Bairdiella chyrsoura (Lacépède) suggest a longer period of time between spawns

INTERSPAWNING INTERVAL OF ALASKAN G. ACULEATUS 2309 at the end of their extended reproductive seasons (Brown-Peterson & Warren, 2001; Waggy, 2004). For G. aculeatus in Alaska, the challenge of producing multiple clutches of eggs every 3 8 days over a relatively short reproductive season after spending up to 7 months under the ice with limited food sources suggests Alaskan fish may allocate most of their energy during the reproductive season to reproductive output rather than somatic growth. Sexually mature nine-spined stickleback Pungitius pungitius (L.) females do not begin the summer growth period until spawning is completed (Griswold & Smith, 1973). Moreover, energy reserves appear to be rapidly depleted during the reproductive season. Bagamian et al. (2004) have shown that female Alaskan G. aculeatus have higher somatic condition at the beginning of the breeding season than at the end, indicating that energy reserves are reduced as spawning activities occur. Thus, the increase in interspawning interval observed as the reproductive season progresses may reflect diminished energy reserves, which result in less available energy for rapid growth of vitellogenic oocytes. Indeed, Bagamian et al. (2004) found a significantly positive relationship between body condition and the presence of a clutch of eggs in Alaskan fish, with the females in better condition more capable of producing a clutch of eggs. The longer interspawning interval later in the season means there is a reduction in reproductive output as the spawning season progresses and implies that estimates of total annual fecundity cannot be made by simply multiplying clutch size by the number of spawning episodes estimated at one time during the reproductive season. Two tactics representing extremes between immediate use and storage of energy resources for reproduction are represented in the dichotomy between income breeders and capital breeders (Drent & Daan, 1980; Jönsson, 1997). Reproductive investment of income breeders is derived from resources gathered during each reproductive period, whereas the reproductive output of capital breeders is supported by energy obtained and stored before the breeding season (Bonnet et al., 1998). Gasterosteus aculeatus meets the high energy demands of egg production through both foraging and depletion of its own energy stores (Wootton, 1976, 1977; Wootton & Evans, 1976). Although G. aculeatus is not a true capital breeder, it may be considered to be so for a single interspawning period because the energy required to produce a clutch of eggs is often so high that energy resource depletion is usually observed (Poizat et al., 1999). This insight is consistent with the data presented here, suggesting that the rate of clutch production in G. aculeatus slows as the interspawning interval increases. With energy income from simultaneous feeding unable to support reproduction, fish appear unable to sustain the initial rate of reproduction as energy stores are depleted. These conclusions are further supported by Wootton s (1994) data showing a direct depletion of lipids and glycogen from the liver and the body of G. aculeatus when the energetic costs of egg production were not met by ingested food. Gasterosteus aculeatus populations worldwide present an interesting opportunity to investigate the relative magnitude of energy storage in relation to its use among populations. The possibility that the rate of clutch production may be in some way related to relative clutch mass (Baker et al., 2008) makes the investigation of interpopulation variation in energy storage and use an intriguing prospect for future study.

2310 N. J. BROWN-PETERSON AND D. C. HEINS We thank H. Martin and E. Birden (Tulane University) for assistance with field sampling in Alaska. E. Birden (Tulane University) also helped with laboratory processing for the 2001 samples. G. Waggy-Grammer (The University of Southern Mississippi, USM) was responsible for much of the histological processing, sectioning and staining and D. Reid (USM) produced the photographic plate in Fig. 1. The field sampling in Alaska was supported by research grants to DCH from the Newcomb College Foundation of Tulane University. References Ali, M. & Wootton, R. J. (1999). Effect of variable food levels on reproductive performance of breeding female three-spined sticklebacks. Journal of Fish Biology 55, 1040 1053. doi: 10.1111/j.1095 8649.1999.tb00739.x Bagamian, K. H., Heins, D. C. & Baker, J. A. (2004). Body condition and reproductive capacity of three-spined stickleback infected with the cestode Schistocephalus solidus. Journal of Fish Biology 64, 1568 1576. doi:10.1111/j.1095 8649.2004.44411.x Baker, J. A. (1994). Life history variation in female threespine stickleback. In The Evolutionary Biology of the Threespine Stickleback (Bell M. A. & Foster S. A., eds), pp. 146 187. Oxford: Oxford University Press. Baker, J. A. & Foster, S. A. (2002). Phenotypic plasticity for life history traits in a stream population of the threespine stickleback, Gasterosteus aculeatus L. Ecology of Freshwater Fish 11, 20 29. doi:10.1034/j.1600-0633.2002.110104.x Baker, J. A., Foster S. A., Heins D. C., Bell, M. A & King, R. W. (1998). Variation in female life-history traits among Alaskan populations of the threespine stickleback, Gasterosteus aculeatus L. (Pisces: Gasterosteidae). Biological Journal of the Linnean Society 63, 141 159. doi:10.1111/j.1095 8312.1998.tb01643.x Baker, J. A., Cresko W. A., Foster, S. A. & Heins, D. C. (2005). Life-history differentiation of benthic and limnetic ecotypes in a polytypic population of threespine stickleback (Gasterosteus aculeatus). Evolutionary Ecology Research 7, 121 131. Baker, J. A., Heins, D. C., Foster, S. A. & King, R. W. (2008). An overview of life-history variation in female threespine stickleback. Behaviour 145, 579 602. Bakker, T. C. M. & Milinski, M. (1991). Sequential female choice and the previous male effect in sticklebacks. Behavioral Ecology and Sociobiology 29, 205 210. Bolduc, F. & FitzGerald, G. J. (1989). The role of selected environmental factors and sex ratio upon egg production in three-spined sticklebacks, Gasterosteus aculeatus. Canadian Journal of Zoology 67, 2013 2020. Bonnet, X., Bradshaw, D. B. & Shine, R. (1998). Capital versus income breeding: an ectothermic perspective. Oikos 83, 333 342. Brewer, S. K., Papoulias, D. M. & Rabeni, C. F. (2006). Spawning habitat associations and selection by fishes in a flow-regulated prairie river. Transactions of the American Fisheries Society 135, 763 778. Brewer, S. K., Rabeni, C. F. & Papoulias, D. M. (2008). Comparing the histology and gonadosomatic index for determining spawning condition of small-bodied riverine fishes. Ecology of Freshwater Fish 17, 54 58. Brown-Peterson, N. J. (2003). The reproductive biology of the spotted seatrout. In The Biology of the Spotted Seatrout (Bortone, S., ed.), pp. 99 133. Boca Raton, FL: CRC Press. Brown-Peterson, N. J. & Warren, J. W. (2001). The reproductive biology of spotted seatrout, Cynoscion nebulosus, along the Mississippi Gulf coast. Gulf of Mexico Science 19, 61 73. Brown-Peterson, N. J., Overstreet, R. M., Lotz, J. M., Franks, J. S. & Burns, K. M. (2001). Reproductive biology of cobia, Rachycentron canadum, from coastal waters of the southern United States. Fisheries Bulletin 99, 15 28. Brown-Peterson, N. J., Peterson, M. S., Nieland, D. L., Murphy, M. D., Taylor, R. G. & Warren, J. R. (2002). Reproductive biology of female spotted seatrout, Cynoscion nebulosus, in the Gulf of Mexico: differences among estuaries? Environmental Biology of Fishes 63, 405 415. Drent, R. H. & Daan, S. (1980). The prudent parent: energetic adjustments in avian breeding. Ardea 68, 225 252.

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2312 N. J. BROWN-PETERSON AND D. C. HEINS Water-Research Investigation Report. 85 4292. Anchorage, AK: US Department of the Interior. Wootton, R. J. (1973). Fecundity of the three-spined stickleback, Gasterosteus aculeatus L. Journal of Fish Biology 5, 683 688. doi:10.1111/j.1095 8649.1973.tb04504.x Wootton, R. J. (1974a). The interspawning interval of female three-spined stickleback, Gasterosteus aculeatus. Journal of Zoology 172, 331 342. Wootton, R. J. (1974b). Changes in the courtship behaviour of female three-spined sticklebacks between spawnings. Animal Behaviour 22, 850 855. Wootton, R. J. (1976). The Biology of the Sticklebacks. London: Academic Press. Wootton, R. J. (1977). Effect of food limitation during the breeding season on the size, body components and egg production of female sticklebacks (Gasterosteus aculeatus). Journal of Animal Ecology 46, 823 834. Wootton, R. J. (1994). Energy allocation of the three-spined stickleback. In The Evolutionary Biology of the Threespine Stickleback (Bell, M. A. & Foster, S. A., eds), pp. 133 143. Oxford: Oxford University Press. Wootton, R. J. & Evans, G. W. (1976). Cost of egg production in the three-spined stickleback (Gasterosteus aculeatus L.). Journal of Fish Biology 8, 385 395. doi:10.1111/j.1095 8649.1976.tb03967.x Electronic Reference Brown-Peterson, N. J., Lowerre-Barbieri S. K., Macewicz, B. J., Saborido-Rey, F., Tomkiewicz, J. & Wyanski, D. W. (2007). An Improved and Simplified Terminology for Reproductive Classification in Fishes, Available at http://www.handle.net/10261/ 11844