We collected guppies as juveniles from Alligator Creek, 30 km southwest of Townsville, Queensland. The use of wild-caught individuals to begin the selection lines ensures that naturally occurring genetic (co)variation is present. We raised the fish for the first selected (parental) generation in 100 litre single sex stock tanks.
We imposed three generations of selection on three selection treatments by selecting (1) directly up and (2) down on male attractiveness and (3) up on female preference for attractive males. We also conducted an unselected control. We use the following two letter abbreviations for the four types of line in the remainder of the manuscript: AT – Up attractiveness; UN – Down attractiveness; PR – Up preference; CO – Control. There were two replicates of each of the four types of line, but due to logistic constraints on the number of fish that we could maintain and measure, the replicate lines were conducted in two blocks. Each block contained one line from each of the four types. We performed the same experimental measures on each block but staggered the blocks by two months.
The dates on which we performed selection in each generation, and the number of individuals that we measured and selected are given in Table . In each generation, selected males and females from a line were placed into a 300 litre tank together to mate and produce offspring. This allows for sexual and other forms of natural selection to operate within lines at this stage, and has both advantages and disadvantages. An advantage over designs in which males and females are randomly paired and mated is that if mate choice is possible, linkage disequilibrium between male attractiveness and female preferences [1
] may be maintained [51
]. This disequilibrium is a crucial element of the genetic architecture of choice and attractiveness, and should be carefully considered when designing selection experiments. The disadvantages of our approach lie in the interpretation of any response (direct or correlated). Gray and Cade [53
] point out that within-line sexual selection may cause an overestimate of the genetic correlation between preference and trait. This is not a problem in our study as we found no evidence of direct or indirect responses in these traits (see results). However, any selection on survival or fecundity within lines may either amplify or attenuate any response to the selection imposed by the researcher, a possibility we address in the discussion.
Table 1 The number of males (M) or females (F) measured (in parentheses) and selected in each generation of selection. The selection treatments include up attractiveness (AT), down attractiveness (UN), up preference for attractive males (PR) and control (CO). (more ...)
Offspring were collected daily and reared at initial densities of ten fry per six-litre tank. At approximately 40 days old, the fry were sexed based on the presence of female egg spots, and separated into single sex tanks. Tanks were covered on three sides with brown paper, and contained both floating and sessile plastic plants to provide refuge from harassment and (in the case of fry) cannibalism. Water was aerated and filtered using air-driven filters under a layer of light brown gravel. The temperature was kept constant at 26°C. A mixture of fluorescent and daylight lighting was used to illuminate the tanks. Throughout the experiment, we fed the fish five times a week on one-day old brine shrimp and twice a week on commercial flake food for tropical fish.
Selecting on attractiveness and preference
Our measures of male attractiveness and female preference were designed to capture these traits for the block as a whole so that preference-display runaways within selection lines do not obscure changes in the treatments relative to other lines within the block. Thus each male was seen by females from every treatment and likewise each female saw males from every treatment.
We measured male attractiveness and female mate choice in behavioural trials in partitioned-aquaria (Figure ). We placed one male into each of the five small compartments, and a naive virgin focal female into the large compartment from where she could observe the five males. In the first generation of selection we randomly assigned one sixth of all males to the PR line, one sixth to the CO line, and one third to each of the AT and UN lines. Selection was applied (if at all) only to the individuals that had been assigned to the appropriate line. In the second and third generations, each choice tank contained one male from each line plus either an extra AT or UN male.
Figure 1 The choice tank used in measuring male attractiveness and female preference. Brown paper covered the side and back walls (bold line). Brown river sand covered the floor of tank. Scored glass separated (solid line) the five small compartments. Transparent (more ...)
Up to twenty choice tanks were used per day during behaviour trials. Tanks were arranged over four rows, orientated towards an observer seated one meter away. During the behavioural trials, two daylight incandescent globes, placed behind the observer, provided lighting. All tanks experienced similar lighting intensities at the water surface (range 1.0–1.9 μmol.m-2.sec-1).
On the evening before a trial, we placed the five males and one female into each choice tank. Observations commenced the following morning between 0700 and 0800 hours and involved scanning all tanks consecutively fifty times. If a female was within one body length of and directly facing the compartment containing a male, we scored his compartment number. A male's attractiveness to a given female was the total number of such "visits" she paid him (maximum possible = 50). Similar partitioned-aquarium measures have been used extensively in studies of guppy mate choice, and attractiveness scores have been shown to significantly predict mating success [15
]. We repeated the behavioural trial over five consecutive days, using a new focal female each day. On two of the five days the female was from the PR line, and the female was from each of the other three lines on one day each.
A male's attractiveness may be influenced by three factors: his actual attractiveness to the females that saw him, the choice tank he was in, and his location within the tank. It is important to control for the latter two factors. Typically, with the choice tank used in this experiment the outer two positions have elevated scores, followed by the next two positions (middle positions) and finally the centre position. To correct for the effect of position, we multiplied the scores of each male by a correction factor. We calculated the correction factor for each week of observations based on the average score recorded at each position in all tanks on all five days in that week.
A preference function is how a female ranks prospective mates based on a specific male trait [54
]. A female's "preference for attractive males" indicates the extent to which a female's choices are consistent with those of her peers [23
]. We estimated a female's preference for attractive males as the slope coefficient of the least-squares regression of how she rated the five males on the mean scores that those males received from the other four females who saw them. A positive slope indicates that a female rated the males in the same way as the other four females, and thus presumably the majority of the population. Furthermore, the larger the positive slope the more strongly the female of interest responded to attractive males. The use of linear regression gradients as estimates of the strengths of selection is valid irrespective of whether the assumptions of linear regression significance tests (e.g. normality) are met [30
]. This method is, however, prone to error because slopes were estimated from only five data points. Furthermore, although there is no autocorrelation in the estimated slopes, the use of the mean of four females' scores as the independent variable (attractiveness) means that there is some nonindependence to the preference estimates within a trial. This nonindependence did not, however, result in significant resemblance between the preference measures taken within a tank in a given week (ANOVA F36,395
= 1.184, P
In the F3 generation, we measured a suite of traits to estimate the direct response of each selected trait and any correlated responses in other potentially correlated traits. We measured male attractiveness, female mate choice, male ornamentation, survival and fecundity. A total of 57 virgin males and 107 virgin females from each selection line per block were used for these terminal measures.
We used two different types of behavioural trials to measure female mate choice and male attractiveness. First, we conducted partitioned-aquaria behavioural trials (as in the selection process) which allow for individual identification of each focal female without direct interactions between males and females. We then used open-aquarium behavioural trials, in which males and females can interact freely and the full range of male courtship and female response behaviours can occur [20
]. In the partitioned-aquaria trials, male attractiveness and female preference for attractive males were measured as described above for selection. We estimated a female's responsiveness as the number of times she was seen with any of the males and discrimination as the coefficient of variation in her number of visits to the five males in the tank.
The open-aquarium behavioural trials were conducted in 100 L aquaria under the same lighting conditions as in the partitioned-aquaria trials. On the night before observations, we placed eight males and eight females into the behavioural tank. Each set of eight males contained two males from each selection treatment. The females on any given day were all from the same selection treatment. We used eight new males and eight new females on each day. Males were individually identified by the observer from their unique colour patterns.
Observations began between 0700 and 0800 hours. We watched each male for a five-minute period in random order, and then for a second five-minute period each in a different random order. Finally, we spent ten minutes scanning the tank, shifting from one male to the other approximately every 30 seconds, to ensure that we observed a total of at least five displays per male. We followed the standard procedures of Houde [20
] in scoring a male's attractiveness as the proportion of his sigmoid displays that elicited at least a "glide" response from a female. We measured female responsiveness as the mean response of females to all males in the trial. Only a single measure of responsiveness could be obtained for each trial, as females cannot be individually distinguished.
We photographed the right side of each male against a white background with a Nikon Coolpix 990 digital camera, including a ruler with millimetre graduations in the picture for calibration. Each male was anaesthetised beforehand with iced water and illuminated dorsally and anteriorly (30° angle of incidence) with low intensity halogen light (Fostec ACE 150 watt light source). We then traced the area of the body, the tail and each colour spot using Measure Master (Version 3.4) digital imaging analysis software. From the tracings, we calculated body area and tail area, and the proportion of the body covered by black, fuzzy black, orange, yellow and iridescent spots. We also counted the of coloured spots on his body.
We conducted short-term adult survival and fecundity trials by placing 50 males from one line (used in the previous attractiveness measures) and 50 naive virgin females from the same line into a 250 litre mating tank. Each day for the next 60 days we collected and counted the number of offspring produced. At the end of the 60-day trial we recorded the number of adults of each sex remaining.
We standardised the measures of traits by block means and standard deviations in order to control for environmental variation among blocks. We then assessed differences between the treatments using nested analysis of variance and one-way analysis of variance.
We estimated realized heritabilities of directly selected traits by applying the standardized form of the breeder's equation (Equation 11.3, ref [27
]). We estimated the response to selection as the difference between the line means for each trait of interest in the F3
generation and the corresponding block's control line means. We then standardised the response to be in units of the control line phenotypic standard deviation for the trait of interest. By standardising the response, we are able to use the intensity of selection, i
(Equation 11.5, ref [27
]), instead of the selection differential, s
, which was considerably more difficult to calculate in this experiment. We calculated i
based on the proportion selected and properties of normal distributions (Appendix A, ref [27
]) and modified our estimate based on the ratio of the selected sex to the other sex (Equation 11.6b, [27
]). We then calculated realized heritabilities from the standardized breeders' equation. Each replicate selection line provides one estimate of the realized heritability, allowing a mean and standard error to be directly estimated for each selection treatment.