Study organism and populations
Heterandria formosa is a live-bearing topminnow in the Poeciliid family that tends to aggregate in aquatic vegetation in shallow water. Their small size and external morphological features indicating sexual maturity make H. formosa an excellent species for life-history studies. In females (Fig. 1A), sexual maturity is visually indicated by the appearance of a small black dot on the anal fin (Fraser and Renton 1940); they mature between 8 and 10 mm (standard length) in approximately 30–40 days. In males (Fig. 1B), sexual maturity is indicated by the development of the gonopodium, a modified anal fin used for sperm transfer (Constanz 1989); they take longer to mature (40–50 days) and mature at a larger body size (10–14 mm) than females.
Parental fish for this experiment were obtained from the Wacissa River (WR) in Jefferson County and Trout Pond (TP) in Leon County. These populations are approximately 50 km apart and genetically isolated (Baer 1998; Soucy and Travis 2003), although molecular data suggest that this isolation is fairly recent (Baer 1998).
We measured offspring performance traits using second generation (F2), lab-reared offspring. Parental stocks of WR and TP fish were initiated with 50 wild caught females and 20 wild caught males from each population. Breeding was carried out in 19L aquaria in a temperature (31°C) and light (14:10 day:night) controlled laboratory. Parental densities were maintained at 5–6 females and 2–3 males per tank during breeding of the parental generation, and tanks were checked weekly for offspring. First generation (F1) offspring were removed and assigned to an F1 density treatment (explained below). Pedigrees were kept for all offspring so that inbreeding in the F1s could be minimized. Only individuals that were born in different tanks were allowed to mate.
Females in the WR F1 breeding tanks were kept at one of two densities in 19L aquaria (either 6 females and 2 males per tank, or 2 females and 2 males per tank). These different maternal densities are within the range of natural densities experienced by WR fish (Leips and Travis 1999) and influence offspring size of both WR and TP females (Leips et al. 2009). All TP F1 breeding tanks had 6 females and 2 males, thus producing large TP offspring. Fish were fed ground Tetramin® (Tetra, Melle, Germany) flake food twice daily ad libitum, so any effects on offspring phenotype from different female densities should have resulted from density differences alone, not from reduced per capita resources. To summarize, three types of F2 offspring were produced: TP offspring from tanks with high female density, WR offspring from tanks with high (WR[H]) density, and WR offspring from tanks with low (WR[L]) density.
Eight treatments were used in a two factor, fractional factorial design (Dunson and Travis 1991; Warner et al. 1991) (Table S1). Treatments evaluated offspring performance (growth rate, age, and body size at maturity) at either low or high density (8 and 32 individuals) in treatments containing either offspring from a single population (TP, or WR(H)) or offspring from both populations in competition (WR[H] with TP, or WR[L] with TP). Thus, there were four single population treatments and four mixed population treatments. Single population treatments evaluated performance of the TP and WR(H) offspring at low and high densities. Two of the mixed population treatments measured the performance of the larger WR(H) offspring when in competition against the smaller TP offspring at both densities. The other two mixed population treatments compared the performance of WR(L) offspring when in competition with TP offspring at both densities. In this case, the offspring were more similar in body size. In each mixed population treatment, TP and WR offspring were mixed 1:1 such that there were either 8 or 32 individuals per unit, matching the total density of the single population treatments.
Execution of the competition experiment
Prior to the initiation of a set of experimental replicates, all F2 offspring were removed from F1 breeding tanks. We then collected offspring for assignment to the different treatment groups for the next 8 days. Thus, the offspring within each treatment were similar, but not identical, in age. A drawback of this procedure is that it minimized our ability to detect age-specific trait differences among treatments because trait measurements were taken weekly. However, our results should not be biased because all treatments used offspring within this range of ages. A subset of offspring from each treatment was photographed to determine initial body size. In the mixed population treatments, we tagged fish from one of the populations with calcein to identify the origin of the fish in subsequent sampling events. WR fish were marked in three of the replicates, TP fish in the remaining two. Calcein binds to calcium in tissues and can be seen in fin rays and scales (Leips et al. 2001). H. formosa were tagged by maintaining individuals for 24 h in a solution of 250 mg/L solution of calcein, buffered to a pH of 5.3 using tris (hydroxymethyl) amino-methane. This dye is retained for at least 5 weeks in the laboratory and had no adverse effects on survival or growth compared to an equal number of unmarked control fish (Leips et al. 2001).
One set of the 8 treatments was started in aquaria in the laboratory at one time, and was then moved to the experimental mesh bags placed in large (850L) cattle watering tanks in an open field at the Florida State University Greenhouse Facility. Each tank contained a mixture of well water and rainwater as well as aquatic vegetation and plankton from Trout Pond. Tank communities were established in five cattle tanks 8 months prior to the initiation of the experiment. Approximately, 2000 mL of suspended plankton from Trout Pond were added to each tank monthly during the course of the experiment.
Each cattle tank contained a set of 8 mesh bags in which the fish from one replicate of each of the 8 treatments were raised. Bags were made from nylon tulle material (1.5 mm mesh size) and framed with glass rods to maintain a rectangular shape. Bags were hung from cords attached to a square PVC frame, and suspended in the tank so that each contained approximately 16L of water (Fig S1). Each bag contained a small amount of submerged vegetation, M. laxum and H. verticillata, to provide cover and substrate for epiphytic growth. H. formosa is omnivorous, and fish in the bags presumably fed on plankton as well as the vegetation and algae in the bags.
Each treatment was replicated 5 times (one replicate per cattle tank). Each time a set of replicates was started, all 8 treatments were initiated in separate 19L aquaria (in 16L of water) in the laboratory at the same time. For the first week, fish in the low-density treatments (cells 1,3,4, and 7 in Table A1, Table S1) were fed twice daily with 10 mg of flake food (an ad libitum amount of food). Fish were fed 15 mg twice daily for the second week, and then 20 mg if individuals were retained in the lab for a third week (see rationale below). High-density treatments (cells 2, 5, 6, and 8 in Table A1, Table S1) received the same ration per aquarium (i.e., one quarter of the food ration per individual). This confounds differences in per capita food amounts and density, but was meant to reflect the effect of increasing density in natural populations. Thus, both resource availability and differences in social environment may have influenced the measured traits.
Fish were moved to a cattle tank when they had grown too large to escape through the mesh bags (2–3 weeks). Each replicate was then censused every week. This sampling interval reduces our power to detect subtle differences among the treatments but was necessary for logistical reasons. Males, females, and immature individuals were sorted by visual inspection and taken to the lab. Calcein tagged and untagged fish were sorted by examining anesthetized individuals under an epifluorescence microscope (Leips et al. 2001). Fish were anesthetized in a low dose of MS-222. In the third sampling period, all calcein-tagged fish were retagged with calcein to ensure that the tag was retained until the next sampling period. Fish in the different categories (tagged and untagged; mature male, mature female, and immature individuals) from all treatments were photographed separately in water-filled bins containing a metric scale and then returned to the cattle tanks. The size of fish in each group was measured from photographs using an image analysis system.
The entire experiment was carried out over the course of a single summer The monitoring period of each replicate ran from the date the replicate was set up until the first female in each bag had reached sexual maturity (or the first TP and WR females had matured in the mixed treatment bags).
The initial sizes of the offspring from all three treatments (TP: n = 29; WR[H]: n = 19, and WR[L]: n = 10) were compared using analysis of variance, with population as the categorical variable (Zar 2009). Tukey's HSD procedure was used for post hoc comparisons of means among the groups. We measured the initial size of fish only in the first replicate to minimize handling of newborn offspring. As the effect of maternal density on offspring size matched that of our previous experiments (Leips et al. 2009), we assumed the maternal density treatment had similar effects on offspring size in the remaining replicates.
We assayed the effects of the different treatments on offspring performance using three traits: the average growth rates of individuals in each group for the first 2 weeks, and the age and size of the first maturing individual of each group from a replicate. Growth rates were calculated only for the first 2 weeks because growth rates of males and females begin to diverge after that time (Parrino, Leips and Travis, unpubl. ms.). Focusing only on growth rate for the first 2 weeks minimizes the effects of chance differences in sex ratio among replicates that could have obscured differences among treatment groups. Average trait values of maturing individuals from each replicate were not used for analyses of age and size at maturity because there was large variation in the proportion of individuals that matured in each replicate. This confounded age-structure with density effects among replicates. In addition, in some cases (particularly in high-density treatments), maturation occurred over a long period of time, skewing the mean and median values differently in the different replicates.
We carried out four analyses of variance for each trait using contrast codes to explore the role of maternal density, offspring density, and population of origin on offspring traits. All analyses (designated as A–D) and associated contrasts are detailed in Table S1. Within any single analysis (A, B, C, or D), there are three contrasts i, ii, and iii. The first two contrasts (i and ii) test for main effects and the third contrast (iii) tests for the interaction between two main effects.
Analysis A uses cells 1, 2, 4, and 6 (Table A1, Table S1): the treatments with offspring from only one stock population (either TP or WR(H)), at two densities, low (N = 8 individuals) or high (4N = 32 individuals). For any dependent variable (growth rate, age, or size at maturity), contrast i tests the effect of quadrupling the density. Contrast ii tests whether TP offspring differ from WR(H). Contrast iii tests whether the effect of quadrupling density differs between stocks, thus testing if the populations are differentially sensitive to the depressant effect of density.
Analysis B uses cells 1, 2, 3, and 5 and only uses data from TP offspring. Contrast i tests whether TP traits differ when they are competing only with other TP fish versus when half the fish are WR(H), given a constant density. Contrast ii tests whether there is an effect of total fish density on TP traits. Contrast iii tests whether the effect of density on TP traits depends on the identity of the competitor with whom the TP fish are competing.
Analysis C asks the same questions as analysis B, but instead focuses on WR(H) offspring and provides complementary inference.
Analysis D uses cells 3, 5, 7, and 8. For this analysis, we focus only on data from the WR fish when in competition with TP fish. Contrast i tests whether WR(H) and WR(L) differ in performance in the presence of TP fish. Contrast ii tests the effect of increased density on WR fish performance. Contrast iii tests whether the density effect differs between WR(H) and WR(L).
We did not compare the competitive ability of TP offspring when pitted against WR(L) and WR(H) offspring. This is because TP fish differed from WR(L) fish in two ways, by population of origin and by the density of the maternal environment. Thus, population of origin and maternal environment would be confounded in this comparison.
A drawback of this analysis design is the number of statistical tests on the same dependent variable; however, this approach has some virtues in that light; within any single analysis (A, B, C, or D), each contrast is independent using Type III SS in the two-way ANOVA.
All statistical analyses (described above) were carried out in SAS ® software, V 9.2, SAS Institute Inc. (Cary, NC) (ANOVA). All dependent variables were log transformed to satisfy assumptions of ANOVA.
We checked for differences in survival among treatments using logistic regression (Proc GENMOD (SAS), binomial distribution, logit link, and scaled deviance options). We found no significant differences in survival among treatments (χ2 = 10.11, P = 0.18) so we did not consider this further. The data on age and size at maturity presented below are only from females because very few males matured in any treatment.