range was unaffected [18]. Yet, vanMarle and Wynn [26] found that in infants the discrimination of auditory events was ratio- dependent also in the small number range, suggesting that infants use a single system of analog magnitude in the auditory domain. Regarding non-human primates, chimpanzees have been shown to increase their reaction times to estimate the number of dots in a large number range but not in a small number range [27], but another study combining the data from four great apes found that numerical ratio was the best performance predictor in the range 0–6 [28]. In support of two-system hypothesis, rhesus monkeys successfully selected the greater group of apple slices with comparison of 1 vs. 2, 2 vs. 3 and 3 vs. 4 [29] while they discriminated between 4 and 8 lemons (1:2 numerical ratio) but not between 4 and 6 (2:3). However two recent studies reported that in rhesus monkeys accuracy was strongly affected by numerical ratio for both small and large quantities, in agreement with the existence of a single non-verbal mechanism over the whole numerical range [16,30]. Several lines of evidence suggest that separate systems for small and large numbers may exist in other vertebrates too. In a recent work on attack/retreat decisions in free-ranging dogs [31], Bonanni and collaborators reported that dogs spontaneously assessed large quantities as noisy magnitudes. In contrast dogs approached aggressively with the same probability when they outnumbered opponents by a 1:2, 2:3 or 3:4 ratio, suggesting that dogs discriminate two small quantities using an object-file mechanism. Among birds, New Zealand robins can discriminate 1 vs. 2, 2 vs. 3 and 3 vs. 4 but not 4 vs. 5 and 5 vs. 6; however these birds can also distinguish between large numbers provided that the numerical ratio is at least 1:2 [32]. Studies on domestic chicks and bees have demonstrated that both species can successfully discriminate 2 vs. 3 while they fail to distinguish the same numerical ratio when two large numbers are presented, such as 4 vs. 6 [33,34]. However, this kind of evidence for two numerical systems may not be conclusive. In some cases there are alternative explanations. For example, if chicks and bees can only represent small numbers, then they will not be able to discriminate 4 from 6. In recent years, numerical competence has been investigated in several teleost species using either operant conditioning or spontaneous preferences [22,23,35,36,37,38]. Two of these studies imply the possibility that separate systems for large and small numbers may exist even in fish. Mosquitofish have been found to discriminate between shoals differing in numerosity when the paired numbers were 1 vs. 2, 2 vs. 3 and 3 vs. 4 but they succeeded with only up to a 1:2 numerical ratio (4 vs. 8 or 8 vs. 16) when they had to discriminate between large numbers [22]. In the second study it has been shown that one-day old guppies can discriminate between small quantities of social companions (,4), showing an inborn ability to elaborate small quantities, while the capacity to discriminate large quantities (4 vs. 8) emerges later, as a consequence of both maturation and social experience. This developmental dissociation indirectly suggests the existence of different systems for small and large quantities also in guppies [37]. Overall, the existence of a phylogenetically shared analog magnitude system appears generally accepted, but authors disagree as to whether a single analog magnitude mechanism accounts for discrimination over the whole numerical range, or a distinct system operates over the small number range. In the present study, we used guppies to test one of the predictions of the hypothesis of two separate systems; according to this hypothesis fishes’ ability to discriminate two large numbers should become more accurate as the difference between them increases while, in discriminating quantities ,4, the performance should not be affected by the numerical ratio. Similar Numerical Systems in Humans and Guppies Guppies were required to choose the more numerous of two available groups of conspecifics. As reference, a group of undergraduate students were required to estimate the larger of two groups of dots while prevented from verbal counting. Both species were presented with the same five numerical ratios (0.25/ 0.33/0.50/0.67/0.75) both within the small number range (1–4) and beyond it. Furthermore, we examined the possible role of learning on performance of the fish, comparing subjects with or without previous experience of social groups. Materials and Methods Ethics Statement The Experiment involving fish complies with all laws of the country (Italy) in which it was performed (D.M. 116192) and was approved by ‘Ministero della Salute’ (permit number: 6726-2011). The experiment with undergraduates was approved by the ethics committee of the Department of General Psychology of University of Padova and was conducted according to the Declaration of Helsinki. Before testing, all participants gave their written consent. Undergraduate experiment In this experiment we adopted a procedure commonly used to measure non-verbal numerical abilities in adults, namely a computerized numerical judgement with sequential presentation of the stimuli [17,36,39]. Undergraduates were required to estimate the larger of two groups of dots while being prevented from using verbal counting. Participants. A total of 18 undergraduate students (three males) between the ages of 18 and 31 (mean age: 21.33) took part in the present study for course credits. The experiments were carried out at the Department of General Psychology, University of Padova. All participants had normal or corrected vision. Stimuli and procedure. The stimuli consisted of 240 pairs of arrays composed of different numbers of black dots. The dots differed in size and appeared in the center of the screen on a white background. The number of dots presented on the screen ranged from 1 to 24. Half of the pairs were controlled for continuous variables (cumulative surface area, density, luminance and overall space occupied by arrays), while the other half were not. Twenty further pairs, with identical features, were created for the initial training phase. The stimuli were displayed on a 17-inch monitor, using E-Prime software, in a darkened room. After a period of dark adaptation, a short familiarization training phase with feedback was presented. The participants initially read the experimental instructions on the screen. A fixation cross then appeared in the center of the screen for 1000 ms, then a group of dots was displayed in the centre of the screen for 150 ms (Fig. 1). Following a 500 ms delay, the participants were shown another group of dots for 150 ms. They were required to estimate which one of the two groups was more numerous by pressing one of two keys on the keyboard. In half of the stimuli the larger group was presented first, in the other half the smaller group was presented first. They were instructed to make their responses as quickly and as accurately as possible. Furthermore, to prevent verbal processing of the stimuli, verbal suppression was introduced during the test by asking the participants to continuously repeat ‘abc’. No feedback was provided during the test. Five numerical ratios were presented (0.25, 0.33, 0.50, 0.67, 0.75) for small (1 vs. 4, 1 vs. 3, 1 vs. 2, 2 vs. 3 and 3 vs. 4) and large (6 vs. 24, 6 vs. 18, 6 vs. 12, 6 vs. 9, 6 vs. 8) numerical contrasts. Reaction time is the common measure of numerical acuity in human studies, especially when, as in our study, participants must PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e31923