Experimental evidence for adaptive phenotypic plasticity in a rock ...

Blackwell Science, LtdOxford, UKBIJBiological Journal of the Linnean Society0024-4066The Linnean Society of London, 2002 77 Original Article N. BOUTON ET AL.

Biological Journal of the Linnean Society, 2002, 77, 185–192. With 1 figure

Experimental evidence for adaptive phenotypic plasticity in a rock-dwelling cichlid fish from Lake Victoria NIELS BOUTON*, FRANS WITTE and JACQUES J. M. VAN ALPHEN Institute of Evolutionary and Ecological Sciences, Leiden University, PO Box 9516, 2300 RA Leiden, the Netherlands Received 26 May 2001; revised and accepted for publication 17 May 2002

We demonstrate adaptive phenotypic plasticity (PP) in Neochromis greenwoodi, a rock-dwelling haplochromine cichlid from Lake Victoria. Adaptive phenotypic plasticity will enhance the chance of survival and reproduction in changing or novel environments. For rock-dwelling cichlids it may contribute to the success of settlement after migration between rocky outcrops or islands, since newly colonized patches of rock may offer different food regimes and different competitors. In our experiments, we simulated such situations by raising fry on different diets and with different competitors. We chose diet treatments in such a way that we could predict the direction of anatomical changes from functional demands. We expected an indirect effect of interspecific competition: selectively removing one prey-type by the competing species would leave N. greenwoodi with the other. We found the predicted phenotypic responses to food in trophic traits of N. greenwoodi. However, in the current experimental set-up we did not find a differentiating effect of species of competitor. We discuss the possible role of PP in allopatric and sympatric speciation. © 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 77, 185−192.

ADDITIONAL KEYWORDS: adaptation − biting-force – feeding − haplochromine − premaxilla − Neochromis greenwoodi.

INTRODUCTION Phenotypic plasticity – an environmentally induced change in the phenotype (Via et al., 1995) – is frequently discussed in relation to speciation (Meyer, 1987; Wimberger, 1991; Day, Pritchard & Schluter, 1994; Robinson & Dukas, 1999; Chapman, Galis & Shinn, 2000; Losos et al., 2000). Phenotypic plasticity (PP) may increase the probability of survival and reproduction in changing or novel environments, perhaps resulting in genetic change through assimilation (Waddington, 1975; West-Eberhard, 1989). Williams (1966) argued that such assimilation leads to canalization and thus to an unfavourable loss of flexible response. However, genetic assimilation can also lead to a shift in the reaction norm without necessarily inducing a reduction in PP (Schlichting & Pigliucci, PLASTICITY IN A CICHLID FROM LAKE VICTORIA

*Corresponding author. Current address: Department of Oceanography and Fisheries, University of the Azores, Cais de Santa Cruz, PT-9901–862, Horta, Azores, Portugal. E-mail: [email protected]

1998). Selection will favour genetic systems that preserve PP when conditions in the new environment fluctuate sufficiently (Slatkin & Lande, 1976). Genetic assimilation that preserves PP will be favourable in the long term; first, because it preadapts populations to possible sudden, permanent changes of the environment that resemble some rare environmental condition in the old situation, and second, because it preserves the possibility for future evolutionary responses (Schlichting & Pigliucci, 1998). Rock-dwelling cichlids of Lake Victoria (the study subject in this paper) may speciate in both allopatry and sympatry at rocky islands and outcrops (Seehausen & van Alphen, 1999; Bouton, 2000). PP may contribute to allopatric speciation by helping individuals to survive and establish populations after migration between islands. Losos et al. (2000) argue that PP may have contributed in this way to radiation of Anolis lizards in the Caribbean. In sympatry, PP may help to establish resource polymorphisms, a possible first step in sympatric speciation (Kawata, 2002). Meyer (1987) and Wimberger (1991) discussed the

© 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 77, 185–192

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possible role of PP in cichlid speciation. Meyer (1987) argued that ‘plasticity may be a form of inertia against speciation’ in Central American cichlids. In line with the traditional view that plastic traits evolve slower than canalized traits (Sultan, 1987) he suggested that the speciose African cichlids are less plastic. Wimberger (1991) taking the contrary view, argued that PP might contribute to speciation in the African great lakes. There have hitherto been few studies on PP in cichlids of these lakes (Witte, Barel & van Oijen, 1997), and most of these concentrate on the molluscivore Astatoreochromis alluaudi (e.g. Hoogerhoud, 1986), a lineage that did not speciate in Lake Victoria. The 100+ species of rock-dwelling haplochromine cichlids from Lake Victoria are an example of an adaptively radiated group (Seehausen et al., 1998). Rockdwelling haplochromines in East African lakes are stenotopic; in these species, PP may be a way to deal with changing local circumstances or with different circumstances at newly colonized patches of rock. These patches of rock often have separate populations (Dorit, 1990; Arnegard et al., 1999), and regularly offer different food regimes and different competitors (Bouton, Seehausen & van Alphen, 1997). In the present study, we investigate phenotypic responses of a rock-dwelling cichlid to different food-types, either as such or as mediated by a competing species. We chose Neochromis greenwoodi Seehausen & Bouton, 1998 for the investigation of phenotypic plasticity. It has the most generalized feeding pattern among rock-dwelling cichlids (Bouton, 1999). In our experiment, N. greenwoodi had to feed in the presence of a more effective suction feeder, Pundamilia nyererei (Witte-Maas & Witte, 1985) or more powerful biter, Neochromis rufocaudalis Seehausen & Bouton, 1998. These three species coexist at many rocky islands and outcrops. Their food in nature varies from predominantly zooplankton and insect larvae in P. nyererei, to both insect larvae and filamentous algae in N. greenwoodi, and mainly filamentous algae in N. rufocaudalis (Bouton et al., 1997). In feeding performance experiments, N. rufocaudalis collected most per bite of a substitute for algae, on average 2.6 times as much as P. nyererei; whereas N. greenwoodi collected 1.7 times as much of the substitute as P. nyererei (Bouton, van Os & Witte, 1998). Feeding on Chaoborus larvae, P. nyererei proved to have the best suction capacity, collecting 1.2 larvae per bout, while N. greenwoodi and N. rufocaudalis collected 1.0 (Bouton et al., 1998). In this study we raised N. greenwoodi on zooplankton and the substitute for algae to test if it was able to improve its anatomy for biting when fed on the substitute for algae and improve its anatomy for suction when fed on zooplankton. We also investigated effects of competition, hypothesizing that each of the

competitors can influence the prey choice of each N. greenwoodi specimen . This in turn would lead to adaptive responses in trophic traits. We chose the treatments in such a way that we could predict the direction of change in trophic traits from functional demands. Variation in these traits was found among rocky island populations of N. greenwoodi, and correlated with diet (Bouton et al., 1999). Thus, our aim was to answer the following questions: (1) whether N. greenwoodi shows phenotypic plasticity in response to different food-types; (2) if so, whether interspecific competition influences prey choice, thereby inducing an adaptive phenotypic response in the competing focal species.

MATERIAL AND METHODS EXPERIMENTAL SET-UP Parent stock of N. greenwoodi was collected at Anchor Island (2°33′20″S, 32°53′5″ W) in the Mwanza Gulf of Lake Victoria. To keep genetic variation to a minimum, we used only one large brood ( c. 110 individuals) taken from parents caught in the wild. Haplochromines are female mouth brooders. Larvae start feeding immediately after their mother releases them, but they cannot feed on algae until about 2 weeks after this time (N. Bouton, pers. obs.). Therefore, we used larvae of this age and randomly divided them between the aquaria. In the aquaria we raised N. greenwoodi either apart or in the presence of N. rufocaudalis or P. nyererei of the same age. Experiments were initiated in March 1993 and lasted 18 months. To avoid phenotypic effects of digging in sand (Witte, 1984), we covered the bottoms of the tanks with plastic sheets instead of sand. We had three different diet treatments: ‘algae’, ‘zooplankton’, and ‘algae + zooplankton’. Filamentous algae (naturally growing on rock boulders in Lake Victoria) were replaced by a substitute referred to as ‘algae’ (Bouton et al., 1998). It was a mixture of commercially available frozen spinach, dry flake food, agar and water. The agar served to fasten the mixture tightly to porous concrete slabs measuring 8 × 4 cm. Slabs with the substitute were replaced twice a day in the tanks. ‘Zooplankton’ consisted of living Daphnia in spring and summer or frozen cubes of Cyclops or Daphnia in the other seasons. Once a week, living Chaoborus larvae were given instead of Cyclops or Daphnia. Close relatives of all these planktonic species are present in Lake Victoria, their densities varying between locations and seasons (Witte et al., 1995). Both living and frozen food were kept in buckets on top of the tanks and inserted into the tanks every 20 min during daytime, using a small pumping unit with a time switch. The cubes defrosted in the buckets


PLASTICITY IN A CICHLID FROM LAKE VICTORIA and the zooplankton was kept in suspension with an airstone. With ‘algae + zooplankton’ fish were fed both food categories. To avoid morphological differences caused by allometry, we tried to balance the diet treatments in such a way that growth of N. greenwoodi would be equal. To this end we did a pilot experiment. To enforce competition, fish were not fed ad libitum; food was only available during short periods and fish were feeding constantly in these periods. We stepwise increased the amount of food (i.e. numbers of slabs with ‘algae’ and numbers of zooplankton cubes and estimated amounts of Daphnia and Chaoborus) during the experiment, taking fish size into account. However, we did not adjust the amount of food between treatments, i.e. treatment groups lagging behind in growth did not receive more food. We investigated PP solely in response to food by raising N. greenwoodi on either ‘algae’ or ‘zooplankton’ (Table 1: control groups A and B). Control groups started with 11 individuals. We investigated effects of food and competition on growth and phenotypic plasticity in competition groups (Table 1: groups C to H). Groups C to H started with 22 fishes, 11 individuals of each of two species. Aquaria C to H had about twice the volume of A and B, and were given a double quantity of food. Each of the six aquaria had N. greenwoodi and one of the other species competing for either ‘zooplankton’ (aquaria C, D), ‘algae’ (aquaria E, F), or for ‘algae + zooplankton’ (aquaria G, H, Table 1). In aquaria with a single food-type we expected differences in growth of N. greenwoodi depending on the

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competing species, i.e. reduced growth if the competitor was better adapted to the food-type than N. greenwoodi and vice versa. In aquaria with two food-types we expected the competitors to influence N. greenwoodi’s prey choice. This could lead to differences in trophic traits depending on the competing species. Although we had 11 individuals of N. greenwoodi in each treatment, the treatments themselves were not replicated. This was a consequence of the high demand on space and time of technicians. Thus it can be argued that tank effects might cause differences between treatments. To avoid these effects we kept other factors constant and all aquaria were connected to one central water-filtering system. Furthermore, we could formulate predictions on the direction of anatomical changes based on functional morphology (see below). The experiments were in compliance with the Dutch animal health care regulations.

MEASUREMENTS, CALCULATIONS, AND PREDICTIONS We used standard length as a measure of size and calculated its mean for each experimental group (Table 1). Standard length (SL) is the distance from the rostral tip of the upper jaw to the origin of the caudal fin (Barel et al., 1977). Following van Leeuwen & Spoor (1987) we calculated biting force of the lower jaw as:

Table 1. Design of the phenotypic plasticity experiment using the focal species N. greenwoodi. Column one lists aquarium and group labels, columns two and three the relative amounts of ‘zooplankton’ (z) and ‘algae’ (a). Column four lists the competing species (if present) with the number of specimens that survived until the end of the experiment in parentheses. All groups started with 11 individuals. Nsurv, number of N. greenwoodi that survived until the end of the experiment; M/F, sex ratio of surviving fishes; M/SL and F/SL, standard length of males and females at the end of the experiment. Each sex was tested to establish whether SL differs between treatments with the same food-type, or between competing species. NS, not significant (ANOVA, P > 0.05) Nsurv Control groups: 200-L aquaria A 1z B 1a

N. gree. only N. gree. only

Competition groups: 375-L aquaria C 2z D 2z E F G H

1z 1z

M/F

M/SL

F/SL

5 4

1/4 0/4

+ N. rufo. (n = 9) + P. nyer. (n = 11)

11 10

5/6 3/7

57.3 ± 2.3 58.7 ± 0.6 NS

53.6 ± 1.6 53.7 ± 2.9 NS

2a 2a

+ N. rufo. (n = 11) + P. nyer. (n = 11)

10 10

4/6 3/7

78.2 ± 2.9 78.0 ± 3.3 NS

71.4 ± 2.3 68.4 ± 2.9 NS

1a 1a

+ N. rufo. (n = 9) + P. nyer. (n = 6)

7 9

1/6 3/6

76.1 75.9 ± 2.8 NS

67.5 ± 2.2 70.1 ± 3.1 NS


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N. BOUTON ET AL. Vm R to Z R to Z = c* * C to D R to T R to T R to Z 1 = c * Vm * * C to D R to T

FT = FM *

where FT is the biting force of the lower jaw, FM is the power that can be exerted by the musculus adductor mandibulae, calculated as a species-specific constant (c) times the physiological cross section of the muscle. The latter is calculated as dry weight (V m) of the muscle divided by fibre length (C to D, Fig. 1A). The constant is omitted in comparisons between treatments, since it is unlikely to differ within a species. R to Z denotes the length of the arm from the centre of rotation (R) perpendicular to a muscle fibre to the point where it crosses this fibre (Z, Fig. 1A), and R to T denotes the length of the lower jaw from centre of rota-

Figure 1. Anatomical models. A. Biting force of the lower jaw (after Van Leeuwen & Spoor, 1987). FT, biting force; R to T, lower jaw length; C to D, muscle fibre length; R to Z, length of arm perpendicular to muscle fibre. B. The premaxilla. b, the angle between ascending and dentigerous arms; A to B, length of the ascending arm.

tion (R) to the tip of the most exterior tooth (T, Fig. 1A). Muscle fibres attach to different points on the posterior extension of the lower jaw, resulting in various values for fibre length (C to D) and arm (R to Z). Therefore, we segmented the muscle surface into six parts and calculated the ratio for each part. The ratio proved to be approximately constant throughout the muscle as both arm and fibre length are decreasing from dorsal to ventral. Thus, we used the average. For direct comparability FT was divided by the square of medial head length (mHL), i.e. the length of the perpendicular line in the medial plane in the triangle formed by the rostral tip of the upper jaw and the most caudal points of the opercula. We used the square of mHL, because biting force is proportional to the cross section of the musculus adductor mandibulae. We measured two aspects of the premaxilla: the relative length of the ascending arm l/mHL and the angle b between the ascending and the dentigerous arms (Fig. 1B, Witte, 1984). For downward suction feeding, most fish species protrude the premaxilla. Protrusion will be enhanced if the ascending arm of the premaxilla (l) is long. In contrast, shortening the ascending arm optimizes transmission of biting force (Otten, 1983). Transmission of biting force is further enhanced by increasing the angle b (Otten, 1983). From the functional and morphological point of view, a high biting force is supposed to be adaptive for fishes feeding on filamentous algae (Barel, 1983; Norton, 1995) and a long ascending arm of the premaxilla adaptive for suction feeding. This was confirmed in feeding experiments on the algae substitute and mosquito larvae (Bouton et al., 1998). Note that trade-offs between suction feeding and biting exist, because these modes of feeding impose conflicting demands on anatomy. We predicted that N. greenwoodi including (more) ‘algae’ in their diet would differ in the following ways from those including (more) ‘zooplankton’: (1) the biting force of the lower jaw would be larger; (2) the angle b between the arms of the premaxilla would be larger, and (3) the ascending arm of the premaxilla would be shorter. To investigate the effect of food alone on the extent of (adaptive) phenotypic plasticity we compared the anatomy between the control groups A and B. To investigate the effects of competition, food, and the interaction between the two, we performed a MANOVA on measures of the six N. greenwoodi groups with the three diet treatments with one of the two competitors (N. rufocaudalis or P. nyererei). In addition we used ANOVA for direct comparison between groups.

RESULTS The control groups (A and B) could not be used for any comparisons since mortality, most likely caused by


PLASTICITY IN A CICHLID FROM LAKE VICTORIA aggression of the dominant fishes, was very high (Table 1). Dominant fishes were seen to chase and bite others, which eventually sustained damage and died. In competition groups consisting of 11 individuals of N. greenwoodi and 11 of another species, aggression was divided over more individuals, and this prevented high mortality. Growth – measured as standard length (SL, Table 1) at the end of the experiment – did not differ between groups of the focal species, i.e. groups that had been raised on the same food-type but with a different competitor. Neochromis greenwoodi raised on ‘algae’ reached the same size when competing with N. rufocaudalis as with P. nyererei, and the same holds for the other diet treatments ‘zooplankton’ and ‘algae + zooplankton’ (Table 1). However, growth of N. greenwoodi differed between diet treatments; the ‘zooplankton’ groups (C and D) remained smaller than the others and did not overlap in size with those including algae in their diet ( ANOVA, P < 0.001, Table 1). We excluded these from the MANOVA that we performed on measures of trophic traits of all competition groups, because we could not distinguish between effects of allometric growth and PP. In the other four groups (E−H) specimens reached similar average sizes (Table 1). In the MANOVA we did not differentiate between sexes, because trophic traits do not differ between sexes in these species (Seehausen et al., 1998). Differences in trophic anatomy between the ‘algae’ and ‘algae + zooplankton’ groups were an effect of food only (MANOVA, Table 2). No effect was found of the competing species or of the interaction between food and competitor (Table 2). To investigate the effect of food, results of measurements on the anatomy of groups with the same food (but different competitor) were joined (E + F and G + H, Table 3). Two out of three anatomical characters (FT/mHL2, b) of N. greenwoodi differed significantly between the treatments ‘algae’ and ‘algae + zooplankton’. In accordance with predictions from functional morphology, the algae groups had a larger

Table 2. Results of MANOVA on the three anatomical characters (FT/mHL2, l/mHL, and β ) of the four N. greenwoodi groups raised with P. nyererei or N. rufocaudalis as a competitor on either ‘algae’ or ‘zooplankton + algae’. See Table 3 for an analysis of the effect of food on anatomy Test for effect

Wilks’ L

F-value

P

food competitor food*competitor

0.756 0.956 0.902

3.34 0.48 1.13

0.032 0.699 0.353

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biting force and a larger angle b between the arms of the premaxilla than the ‘algae + zooplankton’ groups. The length of the ascending arm of the premaxilla did not differ between these groups.

DISCUSSION Mortality is a problem in plasticity experiments, because selective mortality can be an alternative explanation for observed phenotypic differences between treatments. In some studies on PP of cichlids, data on mortality cannot be retrieved, because the number of individuals at the start of the experiment is not given (Meyer, 1987; Wimberger, 1991; Chapman et al., 2000). Except for the control groups and aquarium G, mortality was low in our experiment. Measurements were taken from three specimens (two from aquarium G, one from F) that died before the end of the experiment. Premaxilla measures (biting force of the lower jaw could not be measured due to decay of the muscle tissue) for each of these specimens were around their respective group averages, indicating that mortality was not associated with unfavourable trophic traits. A phenotypic response has been demonstrated in N. greenwoodi; we assume that this is a response to the food offered. However, it can be argued that some other factor caused the observed differences, since individuals with the same treatment were raised together in the same aquarium (the problem of pseudoreplication). There are two arguments against this supposition. First, since we did not find food-bycompetitor-species interactions or differential effects of the competing species, we can independently com-

Table 3. An anatomical comparison between diet treatments: ‘algae’ (A, groups E + F), and ‘algae + zooplankton’ (AZ, groups G + H). Indicated are mean ± SD for standard length (SL) and three trophic traits: biting force of the lower jaw (FT/mHL2), length of the ascending arm of the premaxilla (l/mHL), and angle between the ascending arm and the dentigerous arm of the premaxilla ( β ). P-values are based on two way ANOVAs. Values of the ‘zooplankton’ treatments (Z, groups C + D) are given as well, but not used in any comparison, because of the size difference with the other groups

SL FT/mHL2 (·105) l/mHL (·102) b

A (n = 20)

AZ (n = 16)

P (A–Z)

Z (n = 21)

72.7 ± 5.0 55.4 ± 5.6

70.6 ± 4.2 50.5 ± 8.1

NS