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Individuality in the use of orientation cues by green frogs
Anim. Behav., 28, 1980, 413-425
INDIVIDUALITY IN THE USE OF ORIENTATION CUES BY GREEN FROGS BY K R A I G A D L E R
Section of Neurobiology and Behavior, Cornell University, Langmuir Laboratory, Ithaca, New York 14850 Abstract. The common assumption that test groups are motivationally homogeneous and utilize the same orientation reference cues may not be correct. Green frogs (Rana clamitans) were trained in a circular arena to seek a goalbox located 90 ~ counterclockwise from a lamp. Most frogs learned the task, but an analysis of the training and testing records showed marked individuality in task learning. Some frogs found the goalbox only with the lamp as a cue; others used the goalbox, the goalbox and lamp, or the goalbox and the lamp separately as cues. One individual learned to orient non-randomly to some still-unknown but geographically fixed cue. These observations show that even though frogs can learn a common task, under supposedly identical training conditions they may utilize a diversity of cues. Larger (thus, older) frogs were significantly more consistent in their patterns of movement. Paths of movement that succeeded in reaching the goal tended to be repeated in later tests. Frogs trained to move around a partition to a goal continued that path even when the obstruction was later removed, suggesting the use of a motor memory or kinaesthesia. Standard orientation tests, in which the group was significantly oriented in the expected direction, were shown on closer inspection to consist of frogs moving according to several individually stereotyped factors. Thus, the heterogeneity of individual experimental animals should be more fully taken into consideration in orientation research. amphibians and some other animals is that although the majority in the sample move in the predicted direction, other individuals do not (for example, note responses of animals depicted in Figs. 8-11 in Adler 1976). Since the individuals within test groups are assumed to be motivationally identical, those animals failing to orient at or near the expected bearing are considered to have erred. Such individuals are typically few in number, and the use of statistical techniques permits one to focus on the behaviour of the majority. Hence, the behaviour of so-called 'errant' animals has been virtually ignored. Indeed, the alternative view, that such animals might be motivated differently from the majority or that they might be utilizing different cues, has never been seriously considered. Their orientation, far from being errant, might well be highly appropriate for them. There is a further point to be drawn from this line of thought. It is possible that even among animals orienting as predicted, some individuals orient in that direction for different motivational reasons or utilize different cues to do so. The purpose of this study was to investigate the use of fixed landmarks as orientation cues and to determine the degree of variability in the use of those cues by green frogs (Rana clamitans). Each frog was trained and tested repeatedly in an arena where the position of the cue being tested was rotated randomly, in order to determine
Amphibians use a variety of cues to orient in nature. It is well documented that celestial cues are used (and appropriately time-compensated) and can be perceived both ocularly (Ferguson 1971) and extraoeularly (Adler 1976). Several studies have implicated olfactory cues at least for short-distance movements, both in field (Oldham 1967; Madison 1972) and laboratory (Czeloth 1930; Grubb 1973) experiments. Other studies have suggested the use of geotactic cues (Cummings 1912; Czeloth 1930), memorized motor habits or kinaesthesia (Buytendijk 1918), wind flow (Czeloth 1930) and, in frogs and toads, acoustic cues (Bogert 1960). However, few workers have tested experimentally the use of landmarks as reference cues for orientation by amphibians or, where they did so, they did not attempt to assess separately the use of landmarks from the simultaneous use of other potentially available cues.
In addition, most previous studies purposely tested an individual animal only once or under a given condition only once, to eliminate the possibility of altered behaviour patterns due to earlier testing. Such an approach does not permit an understanding of individual, as opposed to group, behaviour patterns. The usual assumption, at least implicitly, is that a group of animals, whether freshly collected or maintained in the laboratory, is motivationally homogeneous. Yet a common observation in orientation tests with 413
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whether an animal's behaviour was correlated with that cue or instead with some geographically fixed, non-rotated cue or cues. These studies show how frogs use landmarks for orientation and the group heterogeneity and individually stereotyped patterns of these animals. A brief report of some preliminary research on this topic was published previously (Adler 1969).
Materials and Methods Subjects Twelve green frogs, all adult males, were handcollected from a small pond on the campus of the Mountain Lake Biological Station, Giles County, Virginia, on the evening of 26 June 1978. They were kept in a constantly cool room (19 C) where all training and testing was done. While not in the arena, frogs were kept in a partially waterfilled enclosure and exposed to a photoperiod of 14 h (LD 14:10, about 15 tux; incandescent bulbs), beginning at 0600 hours EST, which approximated the natural cycle. Individuals were marked by clipping combinations of the outer two toes on each hind foot. Animals were forcefed raw calves' liver on two occasions during training and testing (10 July, 16 July). Apparatus and Training All training (and testing) was performed in a fiat, level arena surrounded by a circular wall of galvanized metal (60 cm high, about 127 cm in diameter). Eight door openings were spaced equidistantly around the wall, each with an opening at floor level of 10 x 10 cm. Behind each door was a completely enclosed wooden box (10 cm wide, 20 cm high, and 25 cm long) with an opening that snugly fit the flanges of the arena door. The arena was surrounded with opaque black plastic, and an artificial ceiling of black plastic was suspended 150 cm from the floor of the arena. Each animal was trained (and tested) in order at precisely the same time once each day. There were 17 training sessions from 27 June to 19 July, and tests were performed on l, 14, 16, 21, 23, 24, 25, and 26 July; no training or testing was done on 2, 15, 17, 20, or 22 July. All training and testing were conducted between 1400 and 1900 hours EST. During training an incandescent lamp (25 W) was fixed above one of the goalboxes so that its bulb could be seen from any point on the arena floor and thus was available for use as a reference cue. Light intensity was measured with a Tektronix J16 digital photometer, utilizing a J6501 head having a 180 ~ acceptance angle and
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a cosine-corrected diffuser; this instrument is C.I.E. balanced and measures directly in lux. From the centre of the arena, the illuminance at frog's-eye level directly toward the lamp was 31.9 lux; directly toward the wall away from the lamp, 3.2 lux. The position of the lamp corresponded precisely to the position of the goalbox to which frogs were trained, the lamp being 90 ~ clockwise to the position of the box to which training was being made; this position was chosen so that frogs would not simply be trained to move toward or away from the light (and heat) source. During training, a metal liner was placed just inside the arena wall; this 'training liner' had only a single 10 • 10 cm opening that could be rotated and placed in front of the specific goalbox to which a particular frog was being trained. This liner allowed for an open goalbox at any of eight positions around the arena, the exact position chosen for each test and frog being determined separately by a table of random numbers (Rand Corporation 1955). The floor of the arena was covered with brown wrapping paper that was changed after each frog's run; together with wiping of the liner or arena walls, this procedure presumably eliminated the use of odour trails for orientation. Frogs were taken separately from their holding tank just prior to their individual training (or testing) sessions, first placed on dry towelling to remove excess moisture, and then putin the arena centre beneath a translucent, cylindrical plastic container (15 cm diameter, 10 cm high). Frogs could not be seen through the container wall; animals were kept in the containers for 7 rain to overcome effects of handling before the cover was lifted by a pulley-system. Each frog was permitted to move about the arena for 15 min. Throughout this period the animal could enter and leave the open goalbox (as in Fig. 1, session 13), which as a reward contained a moistened sponge on the floor from which the frog could absorb water. However, at 15 min an animal in the training arena was given a noxious stimulus by pushing it into the open goalbox with a stick; this procedure ensured that all animals had contact with the goalbox during each run. Once the animal was in the goalbox (either voluntarily or not), the training liner was turned to close the opening and the animal was then allowed to remain in the box for 5 min before being returned to the common holding tank. Because of this inactive period, sessions of consecutive frogs could be overlapped slightly, allowing a new frog to be introduced every 25 min.
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Fig. 1. Patterns of movement during training. These diagrams depict the movement patterns for a single frog (no. 1) during training sessions 6--15, showing path of movement relative to the goalbox and lamp combination, which was varied randomly in position from session to session (see text). Note the regularity of right-handed or clockwise spirals upon release in centre. For comparative purposes all of the diagrams have been rotated so that the open goalbox is at 90~ but the actual geographicpositions of the goalbox and lamp combination can be determined from the box number of the lamp's position, since the 8 boxes were numbered counterclockwise beginning at 90*. Thus, in session 6 the goalbox was actually at 0~ geographic north. Note that the distribution of points at which the frog touched the side of the arena is concentrated in that half of the arena containing the goalbox (27 versus 7). Details of each frog's movement in the arena were recorded, including its path and duration but also when and where an animal contacted the walls. Occasional frogs jumped to the upper edge of the wall; these were tapped on the snout and pushed back into the arena with no apparent effect on an individual's behaviour.
Testing The random placement of the open goalbox (together with the lamp 90 ~ clockwise to it) was continued during testing as in training. The characteristics of the arena, its floor, the goalboxes, and preparations before release were also identical. However, two additional liners were used, each similar in overall dimensions to the training liner described previously; a 'doorless liner' that was uniform throughout, and a 'falsedoor liner' with a single 10 • 10 cm black plastic piece affixed to it to resemble the door to an open goalbox. With either liner in place, frogs could not actually enter a box to score; therefore, criterion for direction of movement was recorded as either the mean vector of the first five points where an animal tapped against the wall, or that point along the wall where a frog sat motionless for 5 min or more, whichever occurred first. In one test no liners were used at all. During testing, after a frog entered a box (or otherwise
reached criterion) it was re-run immediately after replacing the paper floor and cleaning the arena; animals were thus tested as many times as possible during the 25-min period allotted. In one series of tests the elevated lamp was removed and replaced by three peripheral 100-W incandescent lamps placed equidistant around but below the edge of the arena, thus producing diffuse lighting over the arena. In all statements about the average directional bearings (or mean vector), zero degrees is geographic north.
Procedures for Motor Memory Tests The same arena and release device were used to attempt to replicate with green frogs the experiment of Buytendijk (1918), which demonstrated that toads can repeat memorized patterns of movement. A frog was positioned along the arena wall, about 100~ clockwise to the position of the open goalbox. From its initial position a frog could not directly see the goalbox because of the interposition of an opaque partition beginning flush with the arena wall next to the frog and extending 85 cm into the centre. A lamp was located above the edge of the arena wall, directly opposite the frog's initial position and within its view; the light intensity at the centre of the arena was approximately 32 lux, measured toward the
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lamp. After release, the frog was given 15 min to pass around the end of the incomplete partition and reach the goalbox. Each of 12 frogs was subjected to 18 training sessions on 18 consecutive days; the paper floor covering was changed between each run. After the training was completed, the partition was removed and the path of movement of each frog was recorded during eight subsequent tests.
Statistical Analysis Data were analysed by the Rayleigh test for a non-random distribution and the V-test for concentration in a specific direction (Batschelet 1965, 1972). Results Regularity of Movement during Training The movement pattern of most frogs was individually stereotyped, although that of the group was heterogeneous. For example, one individual's pattern shows, in every instance recorded, a right-handed clockwise spiral upon release and voluntary entry into the open goalbox (Fig. 1, Table I), even though the positions of the open goalbox and lamp were rotated randomly around the arena and the frog's movement was random with respect to the geographically fixed arena itself (Table II). These data suggest that this frog used the position of the lamp or goalbox or both as reference cues in order to locate the goalbox since the goalbo• and lamp were rotated randomly with respect to the arena and any other geographically fixed features in the room. Training records for each frog are given in Table I. Of the total of 12 frogs, 2 (nos. 1 and 6) show significant handedness (one-sample runs test, P < 0.01); 2 others (nos. 4 and 9) that exhibit relatively few changes in hand closely approach significance (P < 0.10). Since it was thought possible that the toe-clipping procedure used to identify individuals might account for the observed left- or right-handed bias, several tests were conducted to evaluate the significance of toe-clipping. In one test, the total number of toes cut (irrespective of side) and in two others, the percentage of right toes clipped was compared to the percentage of right spirals and to the number of changes in handedness. No significant correlations existed (P > 0.05). However, an additional test showed that the group was significantly non-random in handedness (Fig. 2), the larger frogs, by inspection, showing greater right-handedness. A further relationship to size was observed in the consistency
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of the initial turn itself, the larger frogs being significantly more consistent (Fig. 3). In addition, there was a significant correlation between consistency of the initial turn and consistency of voluntarily finding the goalbox (Fig. 4). In this comparison, the consistency of finding the goal was defined as the number of trials necessary before the goal was entered voluntarily three successive times. However, a significant relationship to turning consistency was evident even if 2, 4 or 5 successive voluntary goal entries was used as the criterion. A statistical analysis of the movement patterns of each frog during training (Table II) shows that most individuals moved non-randomly with respect to the goalbox and light, since they ordinarily reached criterion by entering the goalbox voluntarily, even though the position of the open goalbox was rotated randomly in relationship to the fixed arena. For one frog, on one set of tests random selection resulted in alignment of the goalbox and light with the arena; therefore it is not possible to say whether this frog (no. 4) was orienting to the position of the goalbox and light or to the arena during training, although subsequent tests support the former alternative. Frog no. 12 oriented non-randomly to the goalbox and light but usually did not enter the goalbox voluntarily; instead, it typically sat along the arena wall, just clockwise to the position of the light.
Patterns of Movement during Testing During testing, three different combinations of goalbox and light were used in order to assess independently the importance of each cue to a frog in locating the goalbox. Statistical analyses of each individual's movement pattern during the three test conditions are given in Table II. In order to test the ability to find the goalbox's position solely by orienting with reference to the light, a doorless liner was used (two tests, 21 and 23 July). Since frogs tested in such an apparatus could not actually enter a goalbox, the arbitrary criterion for direction of movement was either (i) the mean vector of the first five points at which the animal touched the arena wall, or (ii) the point along the wall where a frog sat motionless for 5 min or longer, whichever occurred first. In the second series of tests a liner with a false goalbox opening was used (two tests, 24 and 25 July) to test for an ability to find the goalbox simply by reference directly to it; the reference light was eliminated and instead the three peripheral lights provided only diffuse lighting. In the final test (two tests, 26 and 27 July) the liner with a false
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Fig. 2. Relationship of turning direction to body size. The percentage of initial right-handed turns is calculated from training sessions 6-17 (see Table I) and does not include those situations where frogs moved into the goalbox along a straight path or those in which they failed to move from the centre or did not contact the side of the arena. Individual frogs are identified by number. The frogs are significantly non-random (%2 = 4.08, P < 0.05, df= 1); by inspection, it appears that the larger frogs show a right-handed bias whereas the smaller frogs appear to show no preference.
Group A: Goalbox alone; or goalbox and light together. Three frogs (nos. 2, 4 and 10) exhibited an orientation pattern indicating that they were capable of locating the goalbox either by directly orienting toward it or by reference to the goalbox and light combination. They were unable, however, to locate the goalbox simply by reference to
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light's position or the goalbox itself would continue to do so, but in addition it was anticipated that some frogs might require both cues together in order to locate the goalbox successfully. Since in these three series of tests the positions of the light or goalbox or both together were rotated randomly with respect to the arena and other geographically fixed reference cues, it was also possible to test whether individual frogs were orienting with respect to some non-rotating cue. On the basis of a statistical analysis of each frog's individual movement pattern under these different test conditions, several groups of movement patterns were identified as described below and in Table II.
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Fig. 3. Relationship of turning consistency to body size. The number of changes in hand of the initial spiral movement pattern is determined from training sessions 6-17 (Table I) and according to the criteria described in Fig. 2. The regression coefficient is significant (r2 = 0.59, P < 0.02, Spearman rank correlation test, twotailed) and suggests that the larger, and presumably older, frogs become more consistent in their movement patterns through experience (see text).
Fig. 4. Relationship of turning consistency to consistency in entry of goalbox. The number of changes in hand of the initial spiral movement is determined as described in Fig. 2. The consistency of entry is determined as the number of trials necessary before the goalbox was entered voluntarily three successive times, during training sessions 1-17. Since two frogs did not voluntarily enter the goalbox even by session 17, these are graphed as being in excess of 20 trials since the earliest they could possibly have met criterion was session 20. The correlation coefficient is significant (r2 = 0.46, P < 0.02, Spearman rank correlation test, two-tailed) and suggests that there is a relationship between the consistency of the initial turning movement and the ability to consistently find the goalbox voluntarily.
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the light; therefore, it is presumably the goal that is used for reference when the light and goalbox are simultaneously present. In all instances where a non-random distribution was observed (Table II), the frogs oriented toward the false opening of the goalbox. Group B: Goalbox and light together. Two frogs (nos. 6 and 9) oriented randomly with respect to the light or the false goalbox when each was presented separately, although both frogs were highly oriented toward the false goalbox when it was presented together with the light cue (Table II), suggesting that the presence of both cues was minimally necessary for their oriented movement.
Group C: Light alone; or goalbox and light together. One frog (no. 1) could not locate the goal directly but did so when the light was present alone or together with a false goal; in the latter instance, it is presumably the light cue that is used for reference when both the light and goalbox are simultaneously present. Group D: Light alone; or goalbox alone; or goalbox and light together. One frog (no. 5) oriented to the goal when either the light or false goal was present separately or together (Table II). Apparently either cue was sufficient for it to find the goalbox. Orientation was more precise when the false goal was present, however (false goal alone, mean vector 88.7 ~ V-test U prob. < 0.001; light alone, 106.1 ~ P < 0.05; false goalbox and light, 91.9 ~ P < 0.01; for these tests, all results were rotated so that the expected direction was at 90~ Group E: Arena or some other geographically fixed cue. Throughout training and testing one animal (no. 7) oriented randomly to the light or false goalbox or to a combination but nonrandomly to the arena itself (Table H). In all training runs this animal consistently oriented to a geographically fixed site between goalboxes 2 and 3 and never once entered a goalbox voluntarily during training or testing (Table 2). These data suggest that this animal was using some fixed cue to position itself within the arena, but the nature of this cue was never determined. Since the arena was level and the paper floor covering was changed after each individual test, it seems unlikely that frog no. 7 used differences in level, topographic features, or odours to orient. It is possible that some peculiarity of the finish of the metal arena wall was distinguished by this frog and used in its orientation, but I was unable to detect any such marking on the wall or any
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feature of the ceiling, the surrounds, or my observing patterns that might have been used for a reference cue. Group F: Indistinguishable patterns. The remaining four frogs (nos. 3, 8, 11 and 12) exhibited patterns of movement that cannot be classified according to the previous groups (Table II), either because they failed to move during tests or because of a small number of completed runs, insufficient for statistical analyses, or both. These observations and statistical tests show that the movement patterns of green frogs are individually stereotyped in most instances, although the individuals in a group vary in terms of the cue or cues used for orientation.
Evidence for Motor Memory Qualitatively, the movement patterns of many frogs provide evidence of the ability to locate a goalbox according to a specific stereotyped memorized motor pattern. For example, both of the frogs that used the lamp position to locate the goalbox (nos. 1 and 5) would move along the arena wall after release, make an abrupt rightangle turn approximately at the site of the goalbox (even though the uniform liner was in place), and proceed to strike the snout against the wall several times before moving on to adjacent positions and repeating the procedure. Even in training, when an open goalbox was available, or during certain tests in which a false goalbox entrance was present, nearly all animals would occasionally move to a place just adjacent to the true or false goalbox, turn abruptly, and then strike against the side as described. The pattern of places at which frogs contacted the arena in this manner is clearly shown in Fig. 1, where the majority of the contacts is in that half of the arena containing the goalbox. Indeed, it was this relationship between position of goalbox and arena wall contacts that led to the use of the mean vector of the first five contacts as one criterion for orientation during the tests in which there was no open goalbox. Further evidence for a motor memory is provided by the experiments in which frogs were trained to move around an opaque partition that was then removed during testing. Each of the 12 frogs trained in this manner exhibited a pattern of movement during testing that at least initially included passing around a non-existent partition (Fig. 5). Only after about five testing sessions (~ = 5.2, range 3-7, n : 12) did the path to the open goalbox become an essentially straight one.
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Group Tests During the course o f individual training and testing, three group tests were conducted according to standard procedures utilized in research on amphibian orientation, in order to provide some measure for comparison. The first o f these was conducted after four days o f training (on 1 July). The training liner was removed, thus exposing all eight goalboxes; between tests o f individual frogs the position o f the light was rotated according to a table o f r a n d o m numbers. The m o v e m e n t o f the frogs in this g r o u p was uniform, although there was a slight but statistically non-significant concentration opposite the light's position (V-test, U = 0.9559, P = 0.39, mean vector 29~ Only three frogs entered the expected goalbox. Subsequently, after a day without training or testing, the frogs were returned to the training schedule for 11 days before two additional group tests were performed. The first o f these (14 July) was performed as before, without a finer. Again group m o v e m e n t was statistically r a n d o m ( U = 1.7071, P = 0.18, mean vector 212 ~ with a slight concentration just clockwise to the light. The second test (16 July) was performed with the doorless uniform liner. The group was concentrated (U ---- 1.9636, P -----0.02, mean vector 131 ~ at a point slightly clockwise to the position of the training goalbox that the frogs would be expected to enter were the doorless liner absent (Fig. 6). However, two animals (nos. 2 and 10) failed to score at all.
Discussion Cues Used in Orientation Test results reveal that the green frogs used a variety o f cues to locate the goalbox. Because the
goalbox, light, or goalbox and light were rotated randomly with respect to the arena and all geographically fixed cues, it is possible to sort out the responses relative to these variables. The study shows that a uniform g r o u p o f frogs trained in a uniform manner to locate a goalbox learn the task but in a non-uniform way. A m o n g the group tested, five categories o f movement can
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L Fig. 6. Group test in arena with a doorless uniform liner. The position of the light (L) is shown, as is the location of the goalbox (GB) the animals were expected to enter if the doorless liner had not been in place. Actually, the positions of the light and goalbox were rotated for each frog's test according to a table of random numbers, but for illustrative purposes are here rotated to identical positions. The arrow depicts the mean vector of the 10 animals that scored; two failed to reach criterion. Statistical analysis would suggest that the group as a whole was significantly oriented in the direction of the goalbox, yet the earlier training and testing records of these frogs reveal that the scores are explained according to factors that vary among individuals (see text).
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be distinguished according to the reference cue or cues utilized (Table II): 1. Goalbox cue alone. 2. Light cue alone. 3. Goalbox and light cue together. 4. Goalbox or light cues, each alone. 5. Some geographically fixed cue. For example, the 'goalbox cue alone' category refers to the pattern exhibited by frogs numbers 2, 4 and 10, which oriented with respect to the goalbox but not to the light; the fact that they oriented also when both goalbox and light were present is interpreted as being due to the presence of the goalbox, the light being superfluous. In category 3, both goalbox and light together were necessary for oriented movement, whereas in category 4 either goalbox or light separately could be so used. Tests in which the frogs oriented to the false goalbox suggest that frogs do not necessarily use a moisture gradient in order to find an open goalbox containing a moist sponge. The sample size is too small to ascribe any special significance to the relative frequencies of the five patterns, but at least one frog oriented with respect to each of the possible cues or combinations of cues, and it is this diversity of learned relationships under supposedly identical training conditions that is so striking. These observations suggest that green frogs can learn a common task, to orient in a specific direction to a goalbox, but may utilize different cues or combinations of cues to do so. In standard orientation tests with amphibians, it is typically assumed that when animals orient in a common direction they have learned the same spatial relationships to the same cues, an assumption that must now be called into question. For example, in Landreth & Fergnson's (1968) similar study with Bufo woodhousei, one cannot properly interpret the movement of individuals with respect to available cues in the absence of previous test records for each animal. To illustrate this point, three orientation tests were performed according to standard techniques but utilizing frogs trained and tested repeatedly and about which much additional information was known. In one test the frogs oriented non-randomly as a group in a particular direction (Fig. 6), but the result can more fully and critically be understood by reference to the training and testing records of individual frogs (Table II). Although the group is significantly concentrated toward the goalbox (V-test, P = 0.02), a closer exami-
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nation suggests different interpretations for the individual behaviours. According to Table II, frogs nos. 6 and 9 orient non-randomly only when both the goalbox and light cues together are available; the absence of a goalbox in this test (uniform liner) suggests that the distribution of these two frogs was, in fact, random. Frogs nos. 1 and 5, on the other hand, were the only frogs conclusively shown to use the light alone as a cue (Table II) and, indeed, both oriented close to the expected position of the goalbox. Under the conditions of this test, significant orientation was not expected in the group (frogs nos. 2, 4, 10) orienting solely by goalbox position. In fact, frogs no. 2 and 10 failed to reach criterion; however, frog no. 4 located the goalbox. Since we know from all previous training and test observations that frog no. 4 was unable to locate the goalbox unless it was actually present, I suspect that its orientation to the goalbox position in this particular test was accidental. Frog no. 7, the one choosing the same geographically fixed position throughout all training and testing, continued to do so in this test and, by chance, was positioned just clockwise to the light. All the remaining frogs (nos. 3, 8, 11 and 12) represented that group described as having indistinguishable patterns. Frog no. 3 scored at the light. Both 8 and 11, however, accurately oriented toward the goalbox, although previous training and testing would not have predicted this behaviour. Finally, frog no. 12, which throughout training sat near the light, did likewise in this test. Such an examination of individual behaviours indicates that, although a test group of animals may show statistically oriented behaviour, this may in part be an artifact owing to fortuitous choices of individuals or to fixed geographical placement of the arena, as was shown in this study. Hence, the individuality and heterogeneity of experimental animals must be more carefully considered in future orientation research. One should not conclude from this study that individual frogs have some natural predisposition to utilize a particular cue, only that among available cues individuals learn different ones. Presumably in another set of trials an individual might, by chance, use a different cue.
Size- (and Age-) Dependent Behaviour Several aspects of the orienting behaviour of green frogs seem to be related to size and thus, by inference, also to age since frogs continue to grow throughout life (Thompson 1942). The rate of growth decreases with age, however, as noted in
ADLER: INDIVIDUALITY IN FROG ORIENTATION Martof's (1956) study of growth in a natural population of Rana clamitans in Michigan. No growth data are available for the green frog population in Virginia from which the experimental animals were taken. However, Martof's (1956) data for Michigan animals can be used roughly to infer relative ages for the Virginia frogs since, as Martof notes, the frogs' growth period is restricted to the warmer months and the growing season at the Virginia locality (due to its higher elevation at about 1180 m) approximates that in Michigan, despite their 5~ difference in latitude. Using Martof's (1956) data, my animals ranged in age from late in their second year to early in their fourth beyond metamorphosis; Martof thought it was unlikely that green frogs exceed a lifespan of five years in the wild. Since all of my experimental animals were adult males collected together from the same small pond and thus had presumably grown up under nearly identical conditions, it seems reasonable to conclude that size differences are positively correlated with age. Larger (thus, older) frogs were more consistent in the direction of their turns after release (Fig. 3). That is, smaller (and younger) frogs were more apt to change the handedness of their turns. Whether this difference results from experience or is due to maturation cannot be concluded from this study. A more curious relationship was the tendency for larger frogs to move in righthanded or clockwise spirals, although the smaller frogs were apparently random with respect to handedness (Fig. 2). Such a tendency has not been previously recorded in orientation studies; yet several investigators have noted the tendency for amphibians to move preferentially left or right in simple mazes (Cummings 1910; Greding 1971). Dill (1977) reported handedness among individual tree frogs in their preference for an escape jumping direction. In Yerkes' (1903) study of green frog behaviour, his training records suggest the existence of individual tendencies to turn left or right in a simple two-choice maze. Two frogs (Yerkes' nos. 1 and 2) show a pronounced right bias, whereas the one other frog (no. 6a) slightly favoured the left. Unfortunately, Yerkes gave no size measurements, and no direct comparison to my study can be made. Furthermore, the tendencies to spiral left or right (as in my study) or move in a maze (as in Yerkes') are not directly comparable, but both studies provide evidence that individual preferences should be given greater consideration in studies of amphibian orientation. As Yerkes demonstrated,
423
however, a frog showing an initial tendency to choose one direction can be conditioned to choose the other after several dozen trials, and such trained preferences can be retained for at least 30 days. Yerkes carefully selected and used 'only animals of exceptional ability' that would move rapidly; hence figures given for his animals may represent extreme rather than average values. In my study, no such selection of animals was undertaken. The basis for the apparent relationship between size (and age) and handedness is not obvious (Fig. 2). This may be a spurious relationship resulting from the small size of the sample or peculiar aspects of the testing apparatus or procedure; yet the existence of other size-dependent aspects in the orientation of green frogs suggests that the relationship may not be artifactual. One explanation might be that in finding a goalbox the larger frogs preferentially moved away from the light source, thus producing a path that, in the majority of cases, would have a clockwise arc. However, in their extensive study of frog phototactic behaviour, Jaeger & Hailman (1973) found that adult green frogs naturally moved toward the light source, even at intensities approximately twice those used in my study. They also found that this response was not significantly affected by adaptational state, time of day or season, or temperature; the response is not significantly different in late tadpoles and adults (Jaeger & Hailman 1976). In my study it was observed that a given frog may initially move away from the light but later move toward it (Fig. 1, session numbers 7, 9, 12 and 14, for example). It is perhaps significant that the most right-handed animals (numbers 1, 5, 6 and 9) are precisely those utilizing the light alone or in combination with the goalbox as orientation reference cues (compare the handedness of frogs in Fig. 2 to the categories of movement patterns listed in Table II). Memorization of Motor Patterns
This study suggests that green frogs use memorized motor patterns, or kinaesthesia, to orient, at least in part. The regularity of the spiral paths (Fig. 1) may indicate memorization of tracks successful in previously locating goals. However, since the visual environment was ordinarily not altered from trial to trial, such stereotypy may instead be the immediate response to the same external stimuli. More convincing evidence for a motor memory is the frogs' habit of striking the wall with their snouts. As noted earlier, when
424
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BEHAVIOUR,
approaching an open goalbox or the false doorway, frogs would often make abrupt turns toward the wall and jump against it several times. Sometimes this would occur after a frog had in fact just passed the goal (for example, Fig. 1, session 10). If paths of movement were oriented by immediately eliciting external stimuli, then one would not expect a frog to pass the goalbox, but if the frogs' movement was directed by some inner memorized pattern and not by external cues, then one might expect them occasionally to pass such external cues. Tests with the removable partition provide additional evidence for a motor memory. Frogs initially trained to move to a goalbox around the incomplete partition continued the same path after removal of the partition, even though the original path was now not the most direct route to the goalbox. Eventually, however, the path straightened so that after about five trials the frogs were entering the box along an essentially straight path (Fig. 5). Replacement of the partition required the frogs to relearn the original detour. Similar results had earlier been obtained for a European toad (Bufo calamita), except that a smaller number of trials was required to achieve a straight path to the goalbox (Buytendijk 1918). Such behaviours might be explained on the basis of memorized movements, although continuation of a detour after removal of the partition may occur because the animals are fixing temporarily upon some intermediate cue (such as the lamp) until the goalbox becomes visible and can be used directly as the cue. However, the gradual adjustment from a detour path to a straight one, in which the path length decreases almost monotonically with successive trials, seems to support more fully the notion of a kinaesthetic sense where the animal gradually readjusts its motor memory. If immediately eliciting external stimuli were used, a straight track to the goalbox would appear as soon as the partition was removed. In any event, a kinaesthetic sense alone is clearly insufficient, and at least one reference cue is required to orient the motor pattern sequence in space.
Acknowledgments This research was supported by a grant from the National Science Foundation (BNS 75-18693). The project was completed during tenure of a Research Fellowship at Mountain Lake Biological Station (NSF GB-3439), and I thank the directors of that station, James L. Riopel and James J. Murray, Jr., for the provision of space,
28,
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equipment and their typical hospitality. I am grateful to Jack P. Hailman, David Ingle, Robert G. Jaeger, Douglas H. Taylor, and George R. Zug for comments on the manuscript. This paper is dedicated, with affection, to Professor Charles F. Walker, University of Michigan.
REFERENCES Adler, K. 1969. How do trained frogs find a goalbox? (abstract). J. Herpetol., 3, 198. Adler, K. 1976. Extraocular photoreception in amphibians. Photochem. Photobiol., 23, 275-298. Batschelet, E. 1965. Statistical Methods for the Analysis of Problems in Animal Orientation and Certain Biological Rhythms. Washington, D.C. : American
Institute of Biological Sciences, Monograph. Batschelet, E. 1972. Recent statistical methods for orientation data. In: Animal Orientation and Navigation (Ed. by S. R. GaUer, K. Schmidt-Koenig, G. J. Jacobs and R. E. Belleville), pp. 61-91. Washington, D.C.: National Aeronautics and Space Administration, puN. 262. Bogert, C. M. 1960. The influence of sound on the behavior of amphibians and reptiles. In: Animal Sounds and Communication (Ed. by W. E. Lanyon and W. N. Tavolga), pp. 137-320. Washington, D.C.: American Institute of Biological Sciences, puN. 7. Buytendijk, F. J. J. 1918. Instinct de la recherche du nid et exp6rience chez les crapauds (Bufo vulgaris et Bufo calamita). Arch. Neerland. Physiol. l'Homme Anita., 2, 1-50.
Cummings, B. F. 1910. The formation of useless habits in two British newts (Molge cristata, Laur., and M. palmata, Schneid.), with observations of their general behaviour. Zoologist, 4th ser., 14, 161-175, 211-222. Cummings, B. F. 1912. Distant orientation in Amphibia. Proe. zool. Soc. London, 1912, 8-19. Czeloth, H. 1930. Untersuchungen tiber die Raumorientierung von Triton. Zeitschr. Vergleich. Physiol., 13, 74-163, Dill, L. M. 1977. 'Handedness' in the Pacific tree frog (Hyla regilla). Can. J. ZooL, 55, 1926-1929. Ferguson, D. E. 1971. The sensory basis of orientation in amphibians. Ann. N.Y. Acad. Sei., 188, 30-36. Greding, E. J., Jr. 1971. Comparative rates of learning in frogs (Ranidae) and toads (Bufonidae). Carib. J. Sci., 11, 203-209. Grubb, J. C. 1973. Olfactory orientation in Bufo woodhousei fowleri, Pseudacris clarki and Pseudacris streckeri. Anita. Behav., 21, 726--732. Jaeger, R. G. & Hailman, J. P. 1973. Effects of intensity on the phototactic responses of adult anuran amphibians: a comparative survey. Z. Tierpsychol., 33, 352-407. Jaeger, R. G. & Hailman, J. P. 1976. Ontogenetic shift of spectral phototactic preferences in anuran tadpoles. J, comp. physiol. Psychol., 90, 930-945. Landreth, H. F. & Ferguson, D. E. 1968. The sun compass of Fowler's toad, Bufo woodhousei fowleri. Behaviour, 30, 27-43. Madison, D. M. 1972. Homing orientation in salamanders: a mechanism involving chemical cues. In: Animal Orientation and Navigation (Ed. by S. R.
ADLER: I N D I V I D U A L I T Y IN F R O G ORIENTATION Galler, K. Schmidt-Koenig, G. J. Jacobs and R. E. Belleville), pp. 485--498. Washington, D.C. : National Aeronautics and Space Administration, publ. 262. Martof, B. S. 1956. Growth and development of the green frog, Rana clamitans, under natural conditions. Am. Midl. Nat., 55, 101-117. Oldham, R. S. 1967. Orienting mechanisms of the green frog, Rana clamitans. Ecology, 48, 477--491. Rand Corporation. 1955. A Million Random Digits
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with 100,000 Normal Deviates. Glencoe, Illinois: Free Press Publ. Thompson, D'A. 1942. On Growth andForm. Cambridge: Cambridge University Press. Yerkes, R. M. 1903. The instincts, habits, and reactions of the frog. PsyehoL Rev. Monogr., 4, 579-638 (also in HarvardPsychol. Stud., 1, 1903). (Received 30 March 1979; revised 15 June 1979; MS. number: A2292)
INDIVIDUALITY IN THE USE OF ORIENTATION CUES BY GREEN FROGS BY K R A I G A D L E R
Section of Neurobiology and Behavior, Cornell University, Langmuir Laboratory, Ithaca, New York 14850 Abstract. The common assumption that test groups are motivationally homogeneous and utilize the same orientation reference cues may not be correct. Green frogs (Rana clamitans) were trained in a circular arena to seek a goalbox located 90 ~ counterclockwise from a lamp. Most frogs learned the task, but an analysis of the training and testing records showed marked individuality in task learning. Some frogs found the goalbox only with the lamp as a cue; others used the goalbox, the goalbox and lamp, or the goalbox and the lamp separately as cues. One individual learned to orient non-randomly to some still-unknown but geographically fixed cue. These observations show that even though frogs can learn a common task, under supposedly identical training conditions they may utilize a diversity of cues. Larger (thus, older) frogs were significantly more consistent in their patterns of movement. Paths of movement that succeeded in reaching the goal tended to be repeated in later tests. Frogs trained to move around a partition to a goal continued that path even when the obstruction was later removed, suggesting the use of a motor memory or kinaesthesia. Standard orientation tests, in which the group was significantly oriented in the expected direction, were shown on closer inspection to consist of frogs moving according to several individually stereotyped factors. Thus, the heterogeneity of individual experimental animals should be more fully taken into consideration in orientation research. amphibians and some other animals is that although the majority in the sample move in the predicted direction, other individuals do not (for example, note responses of animals depicted in Figs. 8-11 in Adler 1976). Since the individuals within test groups are assumed to be motivationally identical, those animals failing to orient at or near the expected bearing are considered to have erred. Such individuals are typically few in number, and the use of statistical techniques permits one to focus on the behaviour of the majority. Hence, the behaviour of so-called 'errant' animals has been virtually ignored. Indeed, the alternative view, that such animals might be motivated differently from the majority or that they might be utilizing different cues, has never been seriously considered. Their orientation, far from being errant, might well be highly appropriate for them. There is a further point to be drawn from this line of thought. It is possible that even among animals orienting as predicted, some individuals orient in that direction for different motivational reasons or utilize different cues to do so. The purpose of this study was to investigate the use of fixed landmarks as orientation cues and to determine the degree of variability in the use of those cues by green frogs (Rana clamitans). Each frog was trained and tested repeatedly in an arena where the position of the cue being tested was rotated randomly, in order to determine
Amphibians use a variety of cues to orient in nature. It is well documented that celestial cues are used (and appropriately time-compensated) and can be perceived both ocularly (Ferguson 1971) and extraoeularly (Adler 1976). Several studies have implicated olfactory cues at least for short-distance movements, both in field (Oldham 1967; Madison 1972) and laboratory (Czeloth 1930; Grubb 1973) experiments. Other studies have suggested the use of geotactic cues (Cummings 1912; Czeloth 1930), memorized motor habits or kinaesthesia (Buytendijk 1918), wind flow (Czeloth 1930) and, in frogs and toads, acoustic cues (Bogert 1960). However, few workers have tested experimentally the use of landmarks as reference cues for orientation by amphibians or, where they did so, they did not attempt to assess separately the use of landmarks from the simultaneous use of other potentially available cues.
In addition, most previous studies purposely tested an individual animal only once or under a given condition only once, to eliminate the possibility of altered behaviour patterns due to earlier testing. Such an approach does not permit an understanding of individual, as opposed to group, behaviour patterns. The usual assumption, at least implicitly, is that a group of animals, whether freshly collected or maintained in the laboratory, is motivationally homogeneous. Yet a common observation in orientation tests with 413
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whether an animal's behaviour was correlated with that cue or instead with some geographically fixed, non-rotated cue or cues. These studies show how frogs use landmarks for orientation and the group heterogeneity and individually stereotyped patterns of these animals. A brief report of some preliminary research on this topic was published previously (Adler 1969).
Materials and Methods Subjects Twelve green frogs, all adult males, were handcollected from a small pond on the campus of the Mountain Lake Biological Station, Giles County, Virginia, on the evening of 26 June 1978. They were kept in a constantly cool room (19 C) where all training and testing was done. While not in the arena, frogs were kept in a partially waterfilled enclosure and exposed to a photoperiod of 14 h (LD 14:10, about 15 tux; incandescent bulbs), beginning at 0600 hours EST, which approximated the natural cycle. Individuals were marked by clipping combinations of the outer two toes on each hind foot. Animals were forcefed raw calves' liver on two occasions during training and testing (10 July, 16 July). Apparatus and Training All training (and testing) was performed in a fiat, level arena surrounded by a circular wall of galvanized metal (60 cm high, about 127 cm in diameter). Eight door openings were spaced equidistantly around the wall, each with an opening at floor level of 10 x 10 cm. Behind each door was a completely enclosed wooden box (10 cm wide, 20 cm high, and 25 cm long) with an opening that snugly fit the flanges of the arena door. The arena was surrounded with opaque black plastic, and an artificial ceiling of black plastic was suspended 150 cm from the floor of the arena. Each animal was trained (and tested) in order at precisely the same time once each day. There were 17 training sessions from 27 June to 19 July, and tests were performed on l, 14, 16, 21, 23, 24, 25, and 26 July; no training or testing was done on 2, 15, 17, 20, or 22 July. All training and testing were conducted between 1400 and 1900 hours EST. During training an incandescent lamp (25 W) was fixed above one of the goalboxes so that its bulb could be seen from any point on the arena floor and thus was available for use as a reference cue. Light intensity was measured with a Tektronix J16 digital photometer, utilizing a J6501 head having a 180 ~ acceptance angle and
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a cosine-corrected diffuser; this instrument is C.I.E. balanced and measures directly in lux. From the centre of the arena, the illuminance at frog's-eye level directly toward the lamp was 31.9 lux; directly toward the wall away from the lamp, 3.2 lux. The position of the lamp corresponded precisely to the position of the goalbox to which frogs were trained, the lamp being 90 ~ clockwise to the position of the box to which training was being made; this position was chosen so that frogs would not simply be trained to move toward or away from the light (and heat) source. During training, a metal liner was placed just inside the arena wall; this 'training liner' had only a single 10 • 10 cm opening that could be rotated and placed in front of the specific goalbox to which a particular frog was being trained. This liner allowed for an open goalbox at any of eight positions around the arena, the exact position chosen for each test and frog being determined separately by a table of random numbers (Rand Corporation 1955). The floor of the arena was covered with brown wrapping paper that was changed after each frog's run; together with wiping of the liner or arena walls, this procedure presumably eliminated the use of odour trails for orientation. Frogs were taken separately from their holding tank just prior to their individual training (or testing) sessions, first placed on dry towelling to remove excess moisture, and then putin the arena centre beneath a translucent, cylindrical plastic container (15 cm diameter, 10 cm high). Frogs could not be seen through the container wall; animals were kept in the containers for 7 rain to overcome effects of handling before the cover was lifted by a pulley-system. Each frog was permitted to move about the arena for 15 min. Throughout this period the animal could enter and leave the open goalbox (as in Fig. 1, session 13), which as a reward contained a moistened sponge on the floor from which the frog could absorb water. However, at 15 min an animal in the training arena was given a noxious stimulus by pushing it into the open goalbox with a stick; this procedure ensured that all animals had contact with the goalbox during each run. Once the animal was in the goalbox (either voluntarily or not), the training liner was turned to close the opening and the animal was then allowed to remain in the box for 5 min before being returned to the common holding tank. Because of this inactive period, sessions of consecutive frogs could be overlapped slightly, allowing a new frog to be introduced every 25 min.
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Fig. 1. Patterns of movement during training. These diagrams depict the movement patterns for a single frog (no. 1) during training sessions 6--15, showing path of movement relative to the goalbox and lamp combination, which was varied randomly in position from session to session (see text). Note the regularity of right-handed or clockwise spirals upon release in centre. For comparative purposes all of the diagrams have been rotated so that the open goalbox is at 90~ but the actual geographicpositions of the goalbox and lamp combination can be determined from the box number of the lamp's position, since the 8 boxes were numbered counterclockwise beginning at 90*. Thus, in session 6 the goalbox was actually at 0~ geographic north. Note that the distribution of points at which the frog touched the side of the arena is concentrated in that half of the arena containing the goalbox (27 versus 7). Details of each frog's movement in the arena were recorded, including its path and duration but also when and where an animal contacted the walls. Occasional frogs jumped to the upper edge of the wall; these were tapped on the snout and pushed back into the arena with no apparent effect on an individual's behaviour.
Testing The random placement of the open goalbox (together with the lamp 90 ~ clockwise to it) was continued during testing as in training. The characteristics of the arena, its floor, the goalboxes, and preparations before release were also identical. However, two additional liners were used, each similar in overall dimensions to the training liner described previously; a 'doorless liner' that was uniform throughout, and a 'falsedoor liner' with a single 10 • 10 cm black plastic piece affixed to it to resemble the door to an open goalbox. With either liner in place, frogs could not actually enter a box to score; therefore, criterion for direction of movement was recorded as either the mean vector of the first five points where an animal tapped against the wall, or that point along the wall where a frog sat motionless for 5 min or more, whichever occurred first. In one test no liners were used at all. During testing, after a frog entered a box (or otherwise
reached criterion) it was re-run immediately after replacing the paper floor and cleaning the arena; animals were thus tested as many times as possible during the 25-min period allotted. In one series of tests the elevated lamp was removed and replaced by three peripheral 100-W incandescent lamps placed equidistant around but below the edge of the arena, thus producing diffuse lighting over the arena. In all statements about the average directional bearings (or mean vector), zero degrees is geographic north.
Procedures for Motor Memory Tests The same arena and release device were used to attempt to replicate with green frogs the experiment of Buytendijk (1918), which demonstrated that toads can repeat memorized patterns of movement. A frog was positioned along the arena wall, about 100~ clockwise to the position of the open goalbox. From its initial position a frog could not directly see the goalbox because of the interposition of an opaque partition beginning flush with the arena wall next to the frog and extending 85 cm into the centre. A lamp was located above the edge of the arena wall, directly opposite the frog's initial position and within its view; the light intensity at the centre of the arena was approximately 32 lux, measured toward the
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lamp. After release, the frog was given 15 min to pass around the end of the incomplete partition and reach the goalbox. Each of 12 frogs was subjected to 18 training sessions on 18 consecutive days; the paper floor covering was changed between each run. After the training was completed, the partition was removed and the path of movement of each frog was recorded during eight subsequent tests.
Statistical Analysis Data were analysed by the Rayleigh test for a non-random distribution and the V-test for concentration in a specific direction (Batschelet 1965, 1972). Results Regularity of Movement during Training The movement pattern of most frogs was individually stereotyped, although that of the group was heterogeneous. For example, one individual's pattern shows, in every instance recorded, a right-handed clockwise spiral upon release and voluntary entry into the open goalbox (Fig. 1, Table I), even though the positions of the open goalbox and lamp were rotated randomly around the arena and the frog's movement was random with respect to the geographically fixed arena itself (Table II). These data suggest that this frog used the position of the lamp or goalbox or both as reference cues in order to locate the goalbox since the goalbo• and lamp were rotated randomly with respect to the arena and any other geographically fixed features in the room. Training records for each frog are given in Table I. Of the total of 12 frogs, 2 (nos. 1 and 6) show significant handedness (one-sample runs test, P < 0.01); 2 others (nos. 4 and 9) that exhibit relatively few changes in hand closely approach significance (P < 0.10). Since it was thought possible that the toe-clipping procedure used to identify individuals might account for the observed left- or right-handed bias, several tests were conducted to evaluate the significance of toe-clipping. In one test, the total number of toes cut (irrespective of side) and in two others, the percentage of right toes clipped was compared to the percentage of right spirals and to the number of changes in handedness. No significant correlations existed (P > 0.05). However, an additional test showed that the group was significantly non-random in handedness (Fig. 2), the larger frogs, by inspection, showing greater right-handedness. A further relationship to size was observed in the consistency
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of the initial turn itself, the larger frogs being significantly more consistent (Fig. 3). In addition, there was a significant correlation between consistency of the initial turn and consistency of voluntarily finding the goalbox (Fig. 4). In this comparison, the consistency of finding the goal was defined as the number of trials necessary before the goal was entered voluntarily three successive times. However, a significant relationship to turning consistency was evident even if 2, 4 or 5 successive voluntary goal entries was used as the criterion. A statistical analysis of the movement patterns of each frog during training (Table II) shows that most individuals moved non-randomly with respect to the goalbox and light, since they ordinarily reached criterion by entering the goalbox voluntarily, even though the position of the open goalbox was rotated randomly in relationship to the fixed arena. For one frog, on one set of tests random selection resulted in alignment of the goalbox and light with the arena; therefore it is not possible to say whether this frog (no. 4) was orienting to the position of the goalbox and light or to the arena during training, although subsequent tests support the former alternative. Frog no. 12 oriented non-randomly to the goalbox and light but usually did not enter the goalbox voluntarily; instead, it typically sat along the arena wall, just clockwise to the position of the light.
Patterns of Movement during Testing During testing, three different combinations of goalbox and light were used in order to assess independently the importance of each cue to a frog in locating the goalbox. Statistical analyses of each individual's movement pattern during the three test conditions are given in Table II. In order to test the ability to find the goalbox's position solely by orienting with reference to the light, a doorless liner was used (two tests, 21 and 23 July). Since frogs tested in such an apparatus could not actually enter a goalbox, the arbitrary criterion for direction of movement was either (i) the mean vector of the first five points at which the animal touched the arena wall, or (ii) the point along the wall where a frog sat motionless for 5 min or longer, whichever occurred first. In the second series of tests a liner with a false goalbox opening was used (two tests, 24 and 25 July) to test for an ability to find the goalbox simply by reference directly to it; the reference light was eliminated and instead the three peripheral lights provided only diffuse lighting. In the final test (two tests, 26 and 27 July) the liner with a false
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Group A: Goalbox alone; or goalbox and light together. Three frogs (nos. 2, 4 and 10) exhibited an orientation pattern indicating that they were capable of locating the goalbox either by directly orienting toward it or by reference to the goalbox and light combination. They were unable, however, to locate the goalbox simply by reference to
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light's position or the goalbox itself would continue to do so, but in addition it was anticipated that some frogs might require both cues together in order to locate the goalbox successfully. Since in these three series of tests the positions of the light or goalbox or both together were rotated randomly with respect to the arena and other geographically fixed reference cues, it was also possible to test whether individual frogs were orienting with respect to some non-rotating cue. On the basis of a statistical analysis of each frog's individual movement pattern under these different test conditions, several groups of movement patterns were identified as described below and in Table II.
Z w k-I-CO
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419
74
76
78
80
82
84
86
SIZE (Snout vent, mm)
Fig. 3. Relationship of turning consistency to body size. The number of changes in hand of the initial spiral movement pattern is determined from training sessions 6-17 (Table I) and according to the criteria described in Fig. 2. The regression coefficient is significant (r2 = 0.59, P < 0.02, Spearman rank correlation test, twotailed) and suggests that the larger, and presumably older, frogs become more consistent in their movement patterns through experience (see text).
Fig. 4. Relationship of turning consistency to consistency in entry of goalbox. The number of changes in hand of the initial spiral movement is determined as described in Fig. 2. The consistency of entry is determined as the number of trials necessary before the goalbox was entered voluntarily three successive times, during training sessions 1-17. Since two frogs did not voluntarily enter the goalbox even by session 17, these are graphed as being in excess of 20 trials since the earliest they could possibly have met criterion was session 20. The correlation coefficient is significant (r2 = 0.46, P < 0.02, Spearman rank correlation test, two-tailed) and suggests that there is a relationship between the consistency of the initial turning movement and the ability to consistently find the goalbox voluntarily.
420
ANIMAL
BEHAVIOUR,
the light; therefore, it is presumably the goal that is used for reference when the light and goalbox are simultaneously present. In all instances where a non-random distribution was observed (Table II), the frogs oriented toward the false opening of the goalbox. Group B: Goalbox and light together. Two frogs (nos. 6 and 9) oriented randomly with respect to the light or the false goalbox when each was presented separately, although both frogs were highly oriented toward the false goalbox when it was presented together with the light cue (Table II), suggesting that the presence of both cues was minimally necessary for their oriented movement.
Group C: Light alone; or goalbox and light together. One frog (no. 1) could not locate the goal directly but did so when the light was present alone or together with a false goal; in the latter instance, it is presumably the light cue that is used for reference when both the light and goalbox are simultaneously present. Group D: Light alone; or goalbox alone; or goalbox and light together. One frog (no. 5) oriented to the goal when either the light or false goal was present separately or together (Table II). Apparently either cue was sufficient for it to find the goalbox. Orientation was more precise when the false goal was present, however (false goal alone, mean vector 88.7 ~ V-test U prob. < 0.001; light alone, 106.1 ~ P < 0.05; false goalbox and light, 91.9 ~ P < 0.01; for these tests, all results were rotated so that the expected direction was at 90~ Group E: Arena or some other geographically fixed cue. Throughout training and testing one animal (no. 7) oriented randomly to the light or false goalbox or to a combination but nonrandomly to the arena itself (Table H). In all training runs this animal consistently oriented to a geographically fixed site between goalboxes 2 and 3 and never once entered a goalbox voluntarily during training or testing (Table 2). These data suggest that this animal was using some fixed cue to position itself within the arena, but the nature of this cue was never determined. Since the arena was level and the paper floor covering was changed after each individual test, it seems unlikely that frog no. 7 used differences in level, topographic features, or odours to orient. It is possible that some peculiarity of the finish of the metal arena wall was distinguished by this frog and used in its orientation, but I was unable to detect any such marking on the wall or any
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feature of the ceiling, the surrounds, or my observing patterns that might have been used for a reference cue. Group F: Indistinguishable patterns. The remaining four frogs (nos. 3, 8, 11 and 12) exhibited patterns of movement that cannot be classified according to the previous groups (Table II), either because they failed to move during tests or because of a small number of completed runs, insufficient for statistical analyses, or both. These observations and statistical tests show that the movement patterns of green frogs are individually stereotyped in most instances, although the individuals in a group vary in terms of the cue or cues used for orientation.
Evidence for Motor Memory Qualitatively, the movement patterns of many frogs provide evidence of the ability to locate a goalbox according to a specific stereotyped memorized motor pattern. For example, both of the frogs that used the lamp position to locate the goalbox (nos. 1 and 5) would move along the arena wall after release, make an abrupt rightangle turn approximately at the site of the goalbox (even though the uniform liner was in place), and proceed to strike the snout against the wall several times before moving on to adjacent positions and repeating the procedure. Even in training, when an open goalbox was available, or during certain tests in which a false goalbox entrance was present, nearly all animals would occasionally move to a place just adjacent to the true or false goalbox, turn abruptly, and then strike against the side as described. The pattern of places at which frogs contacted the arena in this manner is clearly shown in Fig. 1, where the majority of the contacts is in that half of the arena containing the goalbox. Indeed, it was this relationship between position of goalbox and arena wall contacts that led to the use of the mean vector of the first five contacts as one criterion for orientation during the tests in which there was no open goalbox. Further evidence for a motor memory is provided by the experiments in which frogs were trained to move around an opaque partition that was then removed during testing. Each of the 12 frogs trained in this manner exhibited a pattern of movement during testing that at least initially included passing around a non-existent partition (Fig. 5). Only after about five testing sessions (~ = 5.2, range 3-7, n : 12) did the path to the open goalbox become an essentially straight one.
ADLER: INDIVIDUALITY IN FROG ORIENTATION
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Fig. 5. Perpetuation of trained movement patterns during later testing: The first diagram shows the arrangement of the arena during the 18 training periods, in which the frog was allowed to move around an incomplete partition and beneath a lamp (L) to reach an open goalbox. The remaining diagrams show the path of movement of a single frog during the eight subsequent tests in which the partition had been removed. These observations provide evidence that paths of movement are at least partially based on memorized motor patterns.
Group Tests During the course o f individual training and testing, three group tests were conducted according to standard procedures utilized in research on amphibian orientation, in order to provide some measure for comparison. The first o f these was conducted after four days o f training (on 1 July). The training liner was removed, thus exposing all eight goalboxes; between tests o f individual frogs the position o f the light was rotated according to a table o f r a n d o m numbers. The m o v e m e n t o f the frogs in this g r o u p was uniform, although there was a slight but statistically non-significant concentration opposite the light's position (V-test, U = 0.9559, P = 0.39, mean vector 29~ Only three frogs entered the expected goalbox. Subsequently, after a day without training or testing, the frogs were returned to the training schedule for 11 days before two additional group tests were performed. The first o f these (14 July) was performed as before, without a finer. Again group m o v e m e n t was statistically r a n d o m ( U = 1.7071, P = 0.18, mean vector 212 ~ with a slight concentration just clockwise to the light. The second test (16 July) was performed with the doorless uniform liner. The group was concentrated (U ---- 1.9636, P -----0.02, mean vector 131 ~ at a point slightly clockwise to the position of the training goalbox that the frogs would be expected to enter were the doorless liner absent (Fig. 6). However, two animals (nos. 2 and 10) failed to score at all.
Discussion Cues Used in Orientation Test results reveal that the green frogs used a variety o f cues to locate the goalbox. Because the
goalbox, light, or goalbox and light were rotated randomly with respect to the arena and all geographically fixed cues, it is possible to sort out the responses relative to these variables. The study shows that a uniform g r o u p o f frogs trained in a uniform manner to locate a goalbox learn the task but in a non-uniform way. A m o n g the group tested, five categories o f movement can
(GB)
L Fig. 6. Group test in arena with a doorless uniform liner. The position of the light (L) is shown, as is the location of the goalbox (GB) the animals were expected to enter if the doorless liner had not been in place. Actually, the positions of the light and goalbox were rotated for each frog's test according to a table of random numbers, but for illustrative purposes are here rotated to identical positions. The arrow depicts the mean vector of the 10 animals that scored; two failed to reach criterion. Statistical analysis would suggest that the group as a whole was significantly oriented in the direction of the goalbox, yet the earlier training and testing records of these frogs reveal that the scores are explained according to factors that vary among individuals (see text).
422
ANIMAL
BEHAVIOUR,
be distinguished according to the reference cue or cues utilized (Table II): 1. Goalbox cue alone. 2. Light cue alone. 3. Goalbox and light cue together. 4. Goalbox or light cues, each alone. 5. Some geographically fixed cue. For example, the 'goalbox cue alone' category refers to the pattern exhibited by frogs numbers 2, 4 and 10, which oriented with respect to the goalbox but not to the light; the fact that they oriented also when both goalbox and light were present is interpreted as being due to the presence of the goalbox, the light being superfluous. In category 3, both goalbox and light together were necessary for oriented movement, whereas in category 4 either goalbox or light separately could be so used. Tests in which the frogs oriented to the false goalbox suggest that frogs do not necessarily use a moisture gradient in order to find an open goalbox containing a moist sponge. The sample size is too small to ascribe any special significance to the relative frequencies of the five patterns, but at least one frog oriented with respect to each of the possible cues or combinations of cues, and it is this diversity of learned relationships under supposedly identical training conditions that is so striking. These observations suggest that green frogs can learn a common task, to orient in a specific direction to a goalbox, but may utilize different cues or combinations of cues to do so. In standard orientation tests with amphibians, it is typically assumed that when animals orient in a common direction they have learned the same spatial relationships to the same cues, an assumption that must now be called into question. For example, in Landreth & Fergnson's (1968) similar study with Bufo woodhousei, one cannot properly interpret the movement of individuals with respect to available cues in the absence of previous test records for each animal. To illustrate this point, three orientation tests were performed according to standard techniques but utilizing frogs trained and tested repeatedly and about which much additional information was known. In one test the frogs oriented non-randomly as a group in a particular direction (Fig. 6), but the result can more fully and critically be understood by reference to the training and testing records of individual frogs (Table II). Although the group is significantly concentrated toward the goalbox (V-test, P = 0.02), a closer exami-
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nation suggests different interpretations for the individual behaviours. According to Table II, frogs nos. 6 and 9 orient non-randomly only when both the goalbox and light cues together are available; the absence of a goalbox in this test (uniform liner) suggests that the distribution of these two frogs was, in fact, random. Frogs nos. 1 and 5, on the other hand, were the only frogs conclusively shown to use the light alone as a cue (Table II) and, indeed, both oriented close to the expected position of the goalbox. Under the conditions of this test, significant orientation was not expected in the group (frogs nos. 2, 4, 10) orienting solely by goalbox position. In fact, frogs no. 2 and 10 failed to reach criterion; however, frog no. 4 located the goalbox. Since we know from all previous training and test observations that frog no. 4 was unable to locate the goalbox unless it was actually present, I suspect that its orientation to the goalbox position in this particular test was accidental. Frog no. 7, the one choosing the same geographically fixed position throughout all training and testing, continued to do so in this test and, by chance, was positioned just clockwise to the light. All the remaining frogs (nos. 3, 8, 11 and 12) represented that group described as having indistinguishable patterns. Frog no. 3 scored at the light. Both 8 and 11, however, accurately oriented toward the goalbox, although previous training and testing would not have predicted this behaviour. Finally, frog no. 12, which throughout training sat near the light, did likewise in this test. Such an examination of individual behaviours indicates that, although a test group of animals may show statistically oriented behaviour, this may in part be an artifact owing to fortuitous choices of individuals or to fixed geographical placement of the arena, as was shown in this study. Hence, the individuality and heterogeneity of experimental animals must be more carefully considered in future orientation research. One should not conclude from this study that individual frogs have some natural predisposition to utilize a particular cue, only that among available cues individuals learn different ones. Presumably in another set of trials an individual might, by chance, use a different cue.
Size- (and Age-) Dependent Behaviour Several aspects of the orienting behaviour of green frogs seem to be related to size and thus, by inference, also to age since frogs continue to grow throughout life (Thompson 1942). The rate of growth decreases with age, however, as noted in
ADLER: INDIVIDUALITY IN FROG ORIENTATION Martof's (1956) study of growth in a natural population of Rana clamitans in Michigan. No growth data are available for the green frog population in Virginia from which the experimental animals were taken. However, Martof's (1956) data for Michigan animals can be used roughly to infer relative ages for the Virginia frogs since, as Martof notes, the frogs' growth period is restricted to the warmer months and the growing season at the Virginia locality (due to its higher elevation at about 1180 m) approximates that in Michigan, despite their 5~ difference in latitude. Using Martof's (1956) data, my animals ranged in age from late in their second year to early in their fourth beyond metamorphosis; Martof thought it was unlikely that green frogs exceed a lifespan of five years in the wild. Since all of my experimental animals were adult males collected together from the same small pond and thus had presumably grown up under nearly identical conditions, it seems reasonable to conclude that size differences are positively correlated with age. Larger (thus, older) frogs were more consistent in the direction of their turns after release (Fig. 3). That is, smaller (and younger) frogs were more apt to change the handedness of their turns. Whether this difference results from experience or is due to maturation cannot be concluded from this study. A more curious relationship was the tendency for larger frogs to move in righthanded or clockwise spirals, although the smaller frogs were apparently random with respect to handedness (Fig. 2). Such a tendency has not been previously recorded in orientation studies; yet several investigators have noted the tendency for amphibians to move preferentially left or right in simple mazes (Cummings 1910; Greding 1971). Dill (1977) reported handedness among individual tree frogs in their preference for an escape jumping direction. In Yerkes' (1903) study of green frog behaviour, his training records suggest the existence of individual tendencies to turn left or right in a simple two-choice maze. Two frogs (Yerkes' nos. 1 and 2) show a pronounced right bias, whereas the one other frog (no. 6a) slightly favoured the left. Unfortunately, Yerkes gave no size measurements, and no direct comparison to my study can be made. Furthermore, the tendencies to spiral left or right (as in my study) or move in a maze (as in Yerkes') are not directly comparable, but both studies provide evidence that individual preferences should be given greater consideration in studies of amphibian orientation. As Yerkes demonstrated,
423
however, a frog showing an initial tendency to choose one direction can be conditioned to choose the other after several dozen trials, and such trained preferences can be retained for at least 30 days. Yerkes carefully selected and used 'only animals of exceptional ability' that would move rapidly; hence figures given for his animals may represent extreme rather than average values. In my study, no such selection of animals was undertaken. The basis for the apparent relationship between size (and age) and handedness is not obvious (Fig. 2). This may be a spurious relationship resulting from the small size of the sample or peculiar aspects of the testing apparatus or procedure; yet the existence of other size-dependent aspects in the orientation of green frogs suggests that the relationship may not be artifactual. One explanation might be that in finding a goalbox the larger frogs preferentially moved away from the light source, thus producing a path that, in the majority of cases, would have a clockwise arc. However, in their extensive study of frog phototactic behaviour, Jaeger & Hailman (1973) found that adult green frogs naturally moved toward the light source, even at intensities approximately twice those used in my study. They also found that this response was not significantly affected by adaptational state, time of day or season, or temperature; the response is not significantly different in late tadpoles and adults (Jaeger & Hailman 1976). In my study it was observed that a given frog may initially move away from the light but later move toward it (Fig. 1, session numbers 7, 9, 12 and 14, for example). It is perhaps significant that the most right-handed animals (numbers 1, 5, 6 and 9) are precisely those utilizing the light alone or in combination with the goalbox as orientation reference cues (compare the handedness of frogs in Fig. 2 to the categories of movement patterns listed in Table II). Memorization of Motor Patterns
This study suggests that green frogs use memorized motor patterns, or kinaesthesia, to orient, at least in part. The regularity of the spiral paths (Fig. 1) may indicate memorization of tracks successful in previously locating goals. However, since the visual environment was ordinarily not altered from trial to trial, such stereotypy may instead be the immediate response to the same external stimuli. More convincing evidence for a motor memory is the frogs' habit of striking the wall with their snouts. As noted earlier, when
424
ANIMAL
BEHAVIOUR,
approaching an open goalbox or the false doorway, frogs would often make abrupt turns toward the wall and jump against it several times. Sometimes this would occur after a frog had in fact just passed the goal (for example, Fig. 1, session 10). If paths of movement were oriented by immediately eliciting external stimuli, then one would not expect a frog to pass the goalbox, but if the frogs' movement was directed by some inner memorized pattern and not by external cues, then one might expect them occasionally to pass such external cues. Tests with the removable partition provide additional evidence for a motor memory. Frogs initially trained to move to a goalbox around the incomplete partition continued the same path after removal of the partition, even though the original path was now not the most direct route to the goalbox. Eventually, however, the path straightened so that after about five trials the frogs were entering the box along an essentially straight path (Fig. 5). Replacement of the partition required the frogs to relearn the original detour. Similar results had earlier been obtained for a European toad (Bufo calamita), except that a smaller number of trials was required to achieve a straight path to the goalbox (Buytendijk 1918). Such behaviours might be explained on the basis of memorized movements, although continuation of a detour after removal of the partition may occur because the animals are fixing temporarily upon some intermediate cue (such as the lamp) until the goalbox becomes visible and can be used directly as the cue. However, the gradual adjustment from a detour path to a straight one, in which the path length decreases almost monotonically with successive trials, seems to support more fully the notion of a kinaesthetic sense where the animal gradually readjusts its motor memory. If immediately eliciting external stimuli were used, a straight track to the goalbox would appear as soon as the partition was removed. In any event, a kinaesthetic sense alone is clearly insufficient, and at least one reference cue is required to orient the motor pattern sequence in space.
Acknowledgments This research was supported by a grant from the National Science Foundation (BNS 75-18693). The project was completed during tenure of a Research Fellowship at Mountain Lake Biological Station (NSF GB-3439), and I thank the directors of that station, James L. Riopel and James J. Murray, Jr., for the provision of space,
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equipment and their typical hospitality. I am grateful to Jack P. Hailman, David Ingle, Robert G. Jaeger, Douglas H. Taylor, and George R. Zug for comments on the manuscript. This paper is dedicated, with affection, to Professor Charles F. Walker, University of Michigan.
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Institute of Biological Sciences, Monograph. Batschelet, E. 1972. Recent statistical methods for orientation data. In: Animal Orientation and Navigation (Ed. by S. R. GaUer, K. Schmidt-Koenig, G. J. Jacobs and R. E. Belleville), pp. 61-91. Washington, D.C.: National Aeronautics and Space Administration, puN. 262. Bogert, C. M. 1960. The influence of sound on the behavior of amphibians and reptiles. In: Animal Sounds and Communication (Ed. by W. E. Lanyon and W. N. Tavolga), pp. 137-320. Washington, D.C.: American Institute of Biological Sciences, puN. 7. Buytendijk, F. J. J. 1918. Instinct de la recherche du nid et exp6rience chez les crapauds (Bufo vulgaris et Bufo calamita). Arch. Neerland. Physiol. l'Homme Anita., 2, 1-50.
Cummings, B. F. 1910. The formation of useless habits in two British newts (Molge cristata, Laur., and M. palmata, Schneid.), with observations of their general behaviour. Zoologist, 4th ser., 14, 161-175, 211-222. Cummings, B. F. 1912. Distant orientation in Amphibia. Proe. zool. Soc. London, 1912, 8-19. Czeloth, H. 1930. Untersuchungen tiber die Raumorientierung von Triton. Zeitschr. Vergleich. Physiol., 13, 74-163, Dill, L. M. 1977. 'Handedness' in the Pacific tree frog (Hyla regilla). Can. J. ZooL, 55, 1926-1929. Ferguson, D. E. 1971. The sensory basis of orientation in amphibians. Ann. N.Y. Acad. Sei., 188, 30-36. Greding, E. J., Jr. 1971. Comparative rates of learning in frogs (Ranidae) and toads (Bufonidae). Carib. J. Sci., 11, 203-209. Grubb, J. C. 1973. Olfactory orientation in Bufo woodhousei fowleri, Pseudacris clarki and Pseudacris streckeri. Anita. Behav., 21, 726--732. Jaeger, R. G. & Hailman, J. P. 1973. Effects of intensity on the phototactic responses of adult anuran amphibians: a comparative survey. Z. Tierpsychol., 33, 352-407. Jaeger, R. G. & Hailman, J. P. 1976. Ontogenetic shift of spectral phototactic preferences in anuran tadpoles. J, comp. physiol. Psychol., 90, 930-945. Landreth, H. F. & Ferguson, D. E. 1968. The sun compass of Fowler's toad, Bufo woodhousei fowleri. Behaviour, 30, 27-43. Madison, D. M. 1972. Homing orientation in salamanders: a mechanism involving chemical cues. In: Animal Orientation and Navigation (Ed. by S. R.
ADLER: I N D I V I D U A L I T Y IN F R O G ORIENTATION Galler, K. Schmidt-Koenig, G. J. Jacobs and R. E. Belleville), pp. 485--498. Washington, D.C. : National Aeronautics and Space Administration, publ. 262. Martof, B. S. 1956. Growth and development of the green frog, Rana clamitans, under natural conditions. Am. Midl. Nat., 55, 101-117. Oldham, R. S. 1967. Orienting mechanisms of the green frog, Rana clamitans. Ecology, 48, 477--491. Rand Corporation. 1955. A Million Random Digits
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with 100,000 Normal Deviates. Glencoe, Illinois: Free Press Publ. Thompson, D'A. 1942. On Growth andForm. Cambridge: Cambridge University Press. Yerkes, R. M. 1903. The instincts, habits, and reactions of the frog. PsyehoL Rev. Monogr., 4, 579-638 (also in HarvardPsychol. Stud., 1, 1903). (Received 30 March 1979; revised 15 June 1979; MS. number: A2292)