Characterization of tetranucleotide microsatellite loci in a Lake Victorian,haplochromine cichlid fish: a Pundamilia pundamilia × Pundamilia nyererei hybrid

The haplochromine cichlid fishes inhabiting Lake Victoria in East Africa are of great interest to evolutionary biologists. We have isolated and optimized six tetranucleotide and a single dinucleotide locus from a hybrid of the haplochromine cichlid fishes, Pundamilia pundamilia and P. nyererei. Characterization in 18 individuals of P. nyererei from a single wild population revealed between six and 18 alleles per locus, with expected heterozygosity ranging from 0.68 to 0.94. These loci will prove useful for investigations of population structure, and elucidating relationships between closely related species. An additional 26 unoptimized loci have been deposited with GenBank and the MEN database.     

Developmental effects of visual environment on species‐assortative mating preferences in Lake Victoria cichlid fish

Local adaptation can be a potent force in speciation, with environmental heterogeneity leading to niche specialization and population divergence. However, local adaption often requires nonrandom mating to generate reproductive isolation. Population divergence in sensory properties can be particularly consequential in speciation, affecting both ecological adaptation and sexual communication. Pundamilia pundamila and Pundamilia nyererei are two closely related African cichlid species that differ in male coloration, blue vs. red. They co‐occur at rocky islands in southern Lake Victoria, but inhabit different depth ranges with different light environments. The species differ in colour vision properties, and females exert species‐specific preferences for blue vs. red males. Here, we investigated the mechanistic link between colour vision and preference, which could provide a rapid route to reproductive isolation. We tested the behavioural components of this link by experimentally manipulating colour perception – we raised both species and their hybrids under light conditions mimicking shallow and deep habitats – and tested female preference for blue and red males under both conditions. We found that rearing light significantly affected female preference: shallow‐reared females responded more strongly to P. pundamilia males and deep‐reared females favoured P. nyererei males – implying that visual development causally affects mate choice. These results are consistent with sensory drive predictions, suggesting that the visual environment is key to behavioural isolation of these species. However, the observed plasticity could also make the species barrier vulnerable to environmental change: species‐assortative preferences were weaker in females that were reared in the other species’ light condition.     

Preference polymorphism for coloration but no speciation in a population of Lake Victoria cichlids

Female mating preference based on male nuptial coloration hasbeen suggested to be an important source of diversifying selectionin the radiation of Lake Victoria cichlid fish. Initial variationin female preference is a prerequisite for diversifying selection;however, it is rarely studied in natural populations. In clearwater areas of Lake Victoria, the sibling species Pundamiliapundamilia with blue males and Pundamilia nyererei with redmales coexist, intermediate phenotypes are rare, and most femaleshave species-assortative mating preferences. Here, we studya population of Pundamilia that inhabits turbid water wheremale coloration is variable from reddish to blue with most malesintermediate. We investigated male phenotype distribution andfemale mating preferences. Male phenotype was unimodally distributedwith a mode on intermediate color in 1 year and more blue-shiftedin 2 other years. In mate choice experiments with females ofthe turbid water population and males from a clearer water population,we found females with a significant and consistent preferencefor P. pundamilia (blue) males, females with such preferencesfor P. nyererei (red) males, and many females without a preference.Hence, female mating preferences in this population could causedisruptive selection on male coloration that is probably constrainedby the low signal transduction of the turbid water environment.We suggest that if environmental signal transduction was improvedand the preference/color polymorphism was stabilized by negativefrequency-dependent selection, divergent sexual selection mightseparate the 2 morphs into reproductively isolated species resemblingthe clear water species P. pundamilia and P. nyererei.     

Divergent parasite infections in sympatric cichlid species in Lake Victoria

Parasitism has been proposed as a factor in host speciation, as an agent affecting coexistence of host species in species‐rich communities and as a driver of post‐speciation diversification. Young adaptive radiations of closely related host species of varying ecological and genomic differentiation provide interesting opportunities to explore interactions between patterns of parasitism, divergence and coexistence of sympatric host species. Here, we explored patterns in ectoparasitism in a community of 16 fully sympatric cichlid species at Makobe Island in Lake Victoria, a model system of vertebrate adaptive radiation. We asked whether host niche, host abundance or host genetic differentiation explains variation in infection patterns. We found significant differences in infections, the magnitude of which was weakly correlated with the extent of genomic divergence between the host species, but more strongly with the main ecological gradient, water depth. These effects were most evident with infections of Cichlidogyrus monogeneans, whereas the only host species with a strictly crevice‐dwelling niche, Pundamilia pundamilia, deviated from the general negative relationship between depth and parasitism. In accordance with the Janzen–Connell hypothesis, we also found that host abundance tended to be positively associated with infections in some parasite taxa. Data on the Pundamilia sister species pairs from three other islands with variable degrees of habitat (crevice) specialization suggested that the lower parasite abundance of P. pundamilia at Makobe could result from both habitat specialization and the evolution of specific resistance. Our results support influences of host genetic differentiation and host ecology in determining infections in this diverse community of sympatric cichlid species.     

Alteration of tank dimensions reduces male aggression in the swordtail

Intraspecific agonism is a problem in fish culture. This has corresponding fish welfare implications. As the design of the holding container may moderate aggressive behaviour, the aim of this study was to reduce agonistic behaviour by altering tank dimensions without changing the available volume. The study used swordtails, a popular aquarium species in which dominance hierarchies between males are common. The frequency of attacks initiated by dominant males against subordinate males was significantly reduced in tanks with smaller surface area (t₍₁₂₎ = ?3.849, P = 0.002) while the frequency of mating displays, assumed to be a measure of normal behaviour, remained unchanged (t₍₁₂₎ = 1.126, P = 0.282). The time that subordinate males spent feeding was also unaffected (t₍₁₂₎ = ?0.081, P = 0.937) while dominant males reduced the time spent feeding in tanks with smaller surface area (t₍₁₂₎ = ?3.468, P = 0.005). However, this is unlikely to be detrimental to the health of dominant males but may reflect a decreased food requirement through reduced energy expenditure. Alteration of tank dimensions in this way is therefore a simple means for small‐scale facilities and pet stores to reduce agonistic behaviour in swordtails.     

Trade-off between growth rate and aggression in juvenile coho salmon, Oncorhynchus kisutch

In juvenile salmon and trout, there seems to be a positive phenotypic correlation between individual aggression level and growth rate. Aggressive fish are dominant, and they obtain and defend territories, giving them access to good feeding sites. Being aggressive may increase predation risk, and may also carry costs such as increased metabolic demand, with effects on growth. To test the hypothesis that there is a trade-off between individual growth rate and aggression, we mated 12 female coho salmon with two unique males each, creating 24 full-sibling families. Growth of individually marked fish from each family was estimated in a situation where food could not be monopolized. Thereafter, individual fish were tested for mirror-elicited agonistic behaviour. We found significant variation between families in early growth rate, with a high heritability (1.04). There was also significant between-family variation in agonistic behaviour, but activity was generally low and heritability was low (0.25) and not significant. Growth rate and agonistic behaviour were negatively correlated. These results imply that aggressive behaviour has an energetic cost. Copyright 2003 Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour.      

Sexual dimorphism in the tusked frog, Adelotus brevis (Anura:Myobatrachidae): the roles of natural and sexual selection

At least two adaptive processes can lead to the evolution of sexual dimorphism: sexual selection (e.g. male-male combat) or natural selection (e.g. dietary divergence). We investigated the adaptive significance of a distinctive pattern of sexual dimorphism in a south-eastern Australian frog, Adelotus brevis. Male Adelotus grow larger than female conspecifics, have larger heads relative to body size, and have large paired projections (‘tusks’) in the lower jaw. All of these traits are rare among anurans. We quantified the degree of dimorphism in Adelotus, and gathered data on diets and mating systems of this species to evaluate the possible roles of sexual selection and dietary divergence in favoring die evolution of these sexually dimorphic traits. Analysis of prey items in alimentary tracts revealed significant sex differences in prey types. For example, females ate proportionally more arthropods and fewer molluscs than did males. However, this difference is likely to be a secondary consequence of habitat differences between the sexes (due in turn to their different reproductive roles) rather than a selective force for the evolution of sexual dimorphism. Calling males spend their time in moist habitats where pondsnails are abundant, whereas females are more often encountered in the drier arthropod-rich woodlands. A three-year behavioural ecology study on a field population revealed that reproductive males engage in agonistic interactions, with the sexually dimorphic tusks used to attack rivals. Larger body size contributed to male reproductive success. Small males were excluded from calling sites and, among the calling males, larger animals had higher reproductive success (numbers of matings) than did smaller individuals. Hence, the atypical pattern of sexual dimorphism in Adelotus brevis seems to have resulted from sexual selection for larger body size and tusk size in males, in the context of male-male agonistic behaviour, rather than natural selection for ecological divergence between the sexes.     

Parasite-mediated sexual selection and species divergence in Lake Victoria cichlid fish

We investigate the role of parasite-mediated sexual selection in the divergence of two species of Lake Victoria cichlids. Pundamilia pundamilia and Pundamilia nyererei represent a common pattern of male nuptial colour divergence between haplochromine sister species: metallic grey–blue in P. pundamilia and bright yellow and red in P. nyererei . Female mating preferences for different male colours maintain the genetic and phenotypic differentiation of the two species in clear water. Previous work indicated that the red coloration of P. nyererei males, which is subject to directional sexual selection, may be a carotenoid-dependent signal of parasite infestation rate. In the present study, we find a parallel result for P. pundamilia : bright blue males are infected with fewer species of parasites. We also find that parasite infestation rates differ quantitatively between the two species in a way that is consistent with species differences in diet and microhabitat. We conclude that parasite-mediated sexual selection may have contributed to the divergence of female mating preferences between P. pundamilia and P. nyererei , and may currently strengthen reproductive isolation between these species.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 94 , 53–60.     

The role of physiology in the divergence of two incipient cichlid species

Sexual selection on male coloration has been implicated in the evolution of colourful species flocks of East African cichlid fish. During adaptive radiations, animals diverge in multiple phenotypic traits, but the role of physiology has received limited attention. Here, we report how divergence in physiology may contribute to the stable coexistence of two hybridizing incipient species of cichlid fish from Lake Victoria. Males of Pundamilia nyererei (males are red) tend to defeat those of Pundamilia pundamilia (males are blue), yet the two sibling species coexist in nature. It has been suggested that red males bear a physiological cost that might offset their dominance advantage. We tested the hypothesis that the two species differ in oxidative stress levels and immune function and that this difference is correlated with differences in circulating steroid levels. We manipulated the social context and found red males experienced significantly higher oxidative stress levels than blue males, but only in a territorial context when colour and aggression are maximally expressed. Red males exhibited greater aggression levels and lower humoral immune response than blue males, but no detectable difference in steroid levels. Red males appear to trade off increased aggressiveness with physiological costs, contributing to the coexistence of the two species. Correlated divergence in colour, behaviour and physiology might be widespread in the dramatically diverse cichlid radiations in East African lakes and may play a crucial role in the remarkably rapid speciation of these fish.     

Agonistic interactions with asymmetric body size in two adult-age groups of the annual killifish Millerichthys robustus (Miller & Hubbs, 1974)

In this study, the author evaluated two adult age groups of the Mexican rivulus Millerichthys robustus with body size asymmetries to determine the strategies used by an annual killifish during agonistic interactions of different ontogenetic stages. To achieve this goal, the author first characterized the ethogram of agonistic interactions of M. robustus composed of seven behavioural units in males and five behavioural units in females. The author then analysed agonistic interaction strategies used by males and females with body size asymmetries in two groups of different adult ages that represent different ontogenetic stages: (a) just after sexual maturity was reached, at 5 weeks of age, and (b) near natural death, at 24 weeks of age. The agonistic behaviour patterns of M. robustus were compatible with the logic of mutual assessment. Large males had an advantage during their interactions in both age groups, winning all of the encounters. Nonetheless, there was more aggression in 5-week-old fish encounters. In addition, small 24-week-old fish were more aggressive than small 5-week-old fish. These changing strategies may be because of the cost–benefits required during a fight at each ontogenetic stage. In the female encounters, size did not predict winners, as both small and large fish won a similar number of encounters, and some contests remained unresolved regardless of age group. There was a tendency for small females of any age to risk more than males in fights to maintain reproductive fitness.     

Behavioural Divergence in Allopatric and Sympatric Gerbil Species (Rodentia: Gerbillinae)

Southern African gerbils comprise 3 genera, each genus represented by morphologically- and ecologically-similar species. All species are monomorphic, with social systems varying from asocial (Desmodillus auricularis) to colonial (Tatera species). This study compares the behaviour patterns of an allopatric species, T. afra, 2 sympatric species, T. leucogaster and T. brantsii, and a species of a different genus, D. auricularis which is sympatric with T. leucogaster and T. brantsii. Interactions between males and females of the same species were staged and quantified in terms of the frequencies of each of 23 behavioural acts. D. auricularis performed 2 acts, namely “sidle” and “fight” which have not been identified in Gerbillurus or Tatera interactions. Sequences of behaviour were similar in all species, and involved females moving away from males, which followed, sniffed the anogenital region and attempted to mount the females. Females responded by presenting, if receptive, or agonistic behaviour such as aggressive or upright postures. Exploration of the terrarium accounted for a large proportion of the behaviour during the 20 min encounter, and watching the opponent also occurred frequently. Discriminant function analysis successfully separated all 4 species on the basis of behaviour frequencies. Individuals were assigned to the correct species with an accuracy of 80%, indicating that each species exhibits species-specific patterns of behaviour. D. auricularis displayed more agonistic and sexual behaviour and less huddling behaviour than any of the Tatera species. The sympatric species-pair, T. leucogaster and T. brantsii, exhibited more significant difference in behaviour than either of the allopatric species-pairs, T. afra and T. brantsii, and T. afra and T. leucogaster. Divergence in behaviour patterns has occurred in allopatric species, but divergence is more marked in species which are sympatric. It is not known whether divergence between T. leucogaster and T. brantsii occurred during a period of allopatry, or whether divergence has occurred due to selection against hybrids in sympatry.     

Behaviour of Arctic charr Salvelinus alpinus during an induced mating season in captivity: how male relative size influences male behavioural investment and female preference over time

The behaviour of sexually mature Arctic charr Salvelinus alpinus specimens (fifth farm generation) was observed in captivity for four consecutive days. Only agonistic interactions between males of different size were facilitated on the first 2 days, while both agonistic and courtship interactions were possible from the third day up to the end of the experiment. The reliability of behavioural analysis was assessed in order to reduce the possibility of observer errors within the generated datasets. The behavioural investment of big males, small males and females was analysed using general linear models (two‐way repeated measures ANOVAs with time and male size as factors). A peak in the agonistic interactions between males occurred during the first day of interactions, where the agonistic investment of big males was significantly higher than that of small males. This resulted in an increased investment in submissive behaviour by the small males, who consistently performed submissive behaviours from the second day of interactions up to the end of the trial. Big males were found to invest significantly more than small males in courtship behaviours for the duration of the trial. Even though females performed inter‐sexual behaviours towards both big and small males for the entire observation period, female interaction rate towards big males was higher than towards small males. This study suggests that both male investment in mating behaviour and female preference might be related to male characteristics such as body length and that S. alpinus behavioural patterns and mate choice cues might be strongly context‐related and characterized by high levels of behavioural plasticity (i.e. presence–absence of certain behavioural units or potential reversal of a mate choice cue) within the same species. Finally, in light of this, some conservation measures are discussed. In particular, effective management plans should take into account the high level of behavioural plasticity likely to be occurring in this species.     

Fitness correlates of male coloration in a Lake Victoria cichlid fish

Sexual selection by female choice has contributed to the rapidevolution of phenotypic diversity in the cichlid fish speciesflocks of East Africa. Yet, very little is known about the ecologicalmechanisms that drive the evolution of female mating preferences.We studied fitness correlates of male nuptial coloration ina member of a diverse Lake Victoria cichlid lineage, Pundamilianyererei. In this species, male red coloration is subject tointraspecific sexual selection by female mate choice. Male nuptialcoloration plays a critical role also in reproductive isolationbetween this species and the closely related sympatric speciesP. pundamilia. Here, we show that P. nyererei male colorationis carotenoid based, illustrating the potential for honest signalingof individual quality. In a wild population, we found that variationin male coloration was not associated with variation in a setof strongly intercorrelated indicators of male dominance: malesize, territory size, and territory location. Instead, the 2male characters that predominantly determine female choice,territory size and red coloration, may be independent predictorsof male quality: males with bright red coloration and largeterritories had lower parasite infestation rates. As a result,female preferences tended to select against heavily parasitizedmales. Consistent with parasite-mediated sexual selection, maleshad higher and more variable parasite loads than females.     

The cost of living slowly: metabolism,Q10 and repeatability in a South American harvestman

Abstract Resources are usually considered to be limited in caves and underground habitats. Therefore, lower metabolic rates of ectotherms in these environments should be advantageous. The standard metabolic rate (SMR) of the common harvestman Pachylus paessleri Roewer, 1913 (Arachnida, Opiliones) is determined at two ambient temperatures. Repeatability of SMR is estimated by two methods: (i) product–moment correlation on residuals of body mass and (ii) variance components. Estimations of the thermal sensitivity of metabolic rate (individual Q₁₀). SMR of P. paessleri at 20 °C show a mean of 19.01 ± 6.04 and 14.99 ± 3.64 μL CO₂ h^?1 for males and females, respectively. Thermal sensitivity of SMR is 2.11 ± 0.23 with a substantial coefficient of variation of 26.7%. In males, residuals of CO₂ release are significantly repeatable (r_p = 0.61) between measurements realized 5 months apart, which reflects the consistency of this trait over time. As typical soil inhabitants, the harvestman P. paessleri present a lower metabolic rate in comparison with arthropods of similar body mass (e.g. arachnids). Although the coefficient of thermal sensitivity is within of the range reported for arthropods inhabiting Mediterranean zones, it is lower than expected for an organism with such a low SMR. This appears to be the first report of repeatability of metabolic rate in a harvestman species.     

Behavioural and physiological stress responses to handling in wild guanacos

Both ethology and physiology can provide insights about stress conditions in wild animals during handling. Lama guanicoe (guanaco) is the major large herbivore inhabiting arid zones in Argentina. Since 2005, the Payún Matrú Cooperative, a business established and run by native people, has captured and handled guanacos for shearing at Payunia Reserve, Mendoza Province, Argentina, to market the species’ fibre for clothing and other products. The aim of our study was to evaluate the behavioural and physiological stress responses to handling of wild guanacos during these shearing activities. We evaluated stress conditions in guanacos by monitoring the frequency of agonistic behaviour in the holding corrals, following capture, the rate of vocalisation and agonistic behaviour during handling, and by estimating cortisol levels in handled animals. Crowding increased agonistic behaviour; this appeared to be related to dominance conflicts between males in the corrals. Time of corralling increased rate of agonistic behaviour and vocalisations during handling. The rate of behavioural stress responses was negatively related to cortisol levels in males; which may be due to animal fatigue or because behavioural stress responses may help mitigate physiological stress responses. Incorporating our results into animal welfare practices could help reduce stress conditions in wild guanacos. Our results suggest that both behavioural and physiological responses are important measures of stress in handled animals, and that behavioural stress responses cannot be used as a simple surrogate for glucocorticoid levels.     

Invasive <Emphasis Type="Italic">Buddleja davidii</Emphasis> allocates more nitrogen to its photosynthetic machinery than five native woody species

The general-purpose genotype hypothesis and the hypothesis of the evolution of invasiveness predict that invasive species are characterized by particular traits that confer invasiveness. However, these traits are still not well-defined. In this study, ecophysiological traits of eight populations of the invasive shrub Buddleja davidii from a wide range of European locations and five co-occurring native woody species in Germany were compared in a common garden experiment. We hypothesized that the invader has higher resource capture ability and utilization efficiency than the natives. No differences were detected among the eight populations of B. davidii in any of the traits evaluated, indicating that the invader did not evolve during range expansion, thus providing support to the general-purpose genotype hypothesis. The invader showed significantly higher maximum electron transport rate, maximum carboxylation rate, carboxylation efficiency, light-saturated photosynthetic rate (P _max) and photosynthetic nitrogen utilization efficiency (PNUE) than the five natives. Leaf nitrogen content was not significantly different between the invader and the natives, but the invader allocated more nitrogen to the photosynthetic machinery than the natives. The increased nitrogen content in the photosynthetic machinery resulted in a higher resource capture ability and utilization efficiency in the invader. At the same intercellular CO₂ concentration, P _max was significantly higher in the invader than in the natives, again confirming the importance of the higher nitrogen allocation to photosynthesis. The invader reduced metabolic cost by increasing the ratio of P _max to dark respiration rate (R _d), but it did not reduce carbon cost by increasing the specific leaf area and decreasing leaf construction cost. The higher nitrogen allocation to the photosynthetic machinery, P _max, PNUE and P _max/R _d may facilitate B. davidii invasion, although studies involving a wide range of invasive species are needed to understand the generality of this pattern and to fully assess the ecological advantages afforded by these features.     

Squid egg mops provide sensory cues for increased agonistic behaviour between male squid

Males of many species compete for access to females. In order to avoid performing potentially costly agonistic behaviour for their entire adult lives, many group-living males use environmental cues to limit agonistic behaviour to times when it will be of most benefit. Long-finned squid, Loligo pealeii, live less than a year and aggregate in mixed- and single-sex schools. Adults participate in several spawning events, then die. During spawning events, males actively compete for females. Winning males pair with females, which subsequently lay eggs in communal sites on the ocean floor (‘egg mops’). To determine whether males use sensory cues provided by egg mops to regulate agonistic behaviour, we conducted four laboratory experiments. We measured the agonistic responses of pairs of adult males before, during and after exposure to conspecific egg mops. In three experiments, egg mops were manipulated to provide differing sensory stimuli (tactile, water-borne, visual). The addition of conspecific egg mops to tanks of paired male squid dramatically increased agonistic behaviour above control levels within minutes. Male squid were first attracted to the egg mops visually, but contact with the capsules was necessary to increase agonistic behaviour. After initial contact, agonistic behaviour was almost continuous as long as egg mops remained present, even when squid touched the egg mops infrequently. Visual stimuli seemed important in maintaining elevated agonistic behaviour between egg mop touches. When egg mops were removed from the tank, measured agonistic behaviour declined within minutes. When egg mops were added to the tank while covered by an opaque and porous cover that allowed water-borne stimuli to circulate into the tank, squid did not approach the covered egg mop or show increased agonistic behaviour. This result suggests that water-borne stimuli are not sufficient to increase agonistic behaviour. It is unusual for male agonistic behaviour in any species to be increased by contact with fertilized eggs. In this species, however, egg capsules might signal that sexually mature, receptive females are about to lay eggs. Indirect evidence suggests that mating with a female immediately before she lays eggs increases male paternity. If this prediction is true, the presence of egg mops may indicate the optimal time for male squid to establish mating precedence through agonistic bouts. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour.      

Allometric relationship between body mass and aerobic metabolism in zebrafish Danio rerio

The relationship between body mass (M) and metabolic rate was investigated through the assessment of active (R_A) and standard (R_S) metabolic rate at different life stages in zebrafish Danio rerio (5 day‐old larvae, 2 month‐old juveniles and 6 month‐old adults). Scaling exponents and constants were assessed for standard (R_S = 0·273M^0·965 in mgO₂ g^?1 h^?1) and active metabolic rate (R_A = 0·799M^0·926 in mgO₂ g^?1 h^?1). These data provide the basis for further experiments regarding the effects of environmental factors on aerobic metabolism throughout the life cycle of this species.     

Incomplete behavioural isolation and asymmetric female preference in darter sister species (Percidae: Etheostoma)

In this study, female preferences and behavioural isolation were estimated in a pair of allopatric sister species, Etheostoma duryi and Etheostoma flavum. Dichotomous mate preference trials were conducted to determine whether females prefer to associate with conspecific over heterospecific males and free‐spawning assays were conducted to determine whether those preferences translated into behavioural isolation. Dichotomous mate choice trials revealed asymmetric female preference, as female E. flavum preferred conspecific males, whereas female E. duryi showed no preference. Free‐spawning assays indicated that behavioural isolation remains incomplete between E. duryi and E. flavum (I_B = 0·19). In addition to female mating preferences, male behaviour also appeared to influence mating outcomes as male E. flavum consistently courted conspecific females more often in free‐spawning assays whereas male E. duryi did not. The data therefore suggest that despite marked divergence in male nuptial colour, divergence in female preferences between these species may not be sufficient to maintain species boundaries upon secondary contact. These results contrast with similar work in a sympatric darter species pair and may be explained by considering the contributions of reinforcement and differences in colour pattern as well as colour value.