Water Ph During Early Development Influences Sex Ratio and Male

Zoology 116 (2013) 139–143

Contents lists available at SciVerse ScienceDirect

Zoology

journa l homepage: www.elsevier.com/locate/zool

Water pH during early development inﬂuences sex ratio and male morph in a

West African cichlid ﬁsh, Pelvicachromis pulcher

∗

Adam R. Reddon , Peter L. Hurd

Department of Psychology, University of Alberta, Edmonton, Alberta, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Environmental sex determination (ESD) is one of the most striking examples of phenotypic plasticity. Indi-

Received 27 June 2012

viduals from species that exhibit ESD can develop as either males or females depending on the particular

Received in revised form 31 October 2012

environmental conditions they experience during early development. In ﬁsh, ESD species often show a

Accepted 22 November 2012

relatively subtle effect of environment, resulting in a substantial number of both sexes being produced in

Available online 7 March 2013

both male- and female-biasing conditions, rather than the unisex clutches that are typical of many rep-

tiles. This less dramatic form of ESD allows the opportunity to study the effects of sexual differentiation

Keywords:

on within-sex variation in behavior and morphology by comparing same-sex individuals produced in

Pelvicachromis pulcher

male- and female-biasing conditions. Here, we conﬁrm that sex determination in the West African cich-

Environmental sex determination

lid, Pelvicachromis pulcher, is inﬂuenced by pH during early development. We show that pH also affects

Phenotypic plasticity

pH treatment the ratio of two alternative male reproductive types with the polygynous morph being overproduced in

Aggression male-biasing conditions and the monogamous male morph being overproduced in female-biasing condi-

tions. Our results suggest that the sexual differentiation process may be an important force in maintaining

individual variation in behavior and reproductive tactics.

1. Introduction often strongly deterministic and small changes in the environment

can lead to single-sex clutches (Bull and Vogt, 1979; Janzen and

Phenotypic plasticity is the ability for a given genotype to gener- Paukstis, 1991). In ﬁsh, however, ESD is typically subtler, and a sub-

ate alternative phenotypes depending on the environment in which stantial number of the minority sex are normally produced (Römer

that phenotype is expressed (Schlichting, 1986; West-Eberhard, and Beisenherz, 1996; Devlin and Nagahama, 2002). This more

1989; Thompson, 1991). Phenotypic plasticity is adaptive in vari- restricted form of ESD in which the sex of the developing animal is

able environments in that it allows the phenotype to be matched to inﬂuenced, but not completely determined, by the ambient envi-

the ambient environmental conditions, permitting greater ﬂexibil- ronment allows the unique opportunity to investigate within-sex

ity in the face of environmental uncertainty (Pigliucci, 1996, 2001, effects of phenotypic plasticity. The sexual differentiation process,

2005; Dingemanse et al., 2009). guided by the developmental environment, may generate differ-

Environmentally inﬂuenced sex determination (ESD) is the pro- ences between the phenotypes of same-sex animals produced in

cess by which the sex of a developing animal is inﬂuenced or different environments (Crews et al., 1998; Rhen and Crews, 2002;

determined by the environmental conditions in which that animal Crews and Groothuis, 2005). For example, females that develop as

develops (Bull and Vogt, 1979; Kraak and Pen, 2002). ESD repre- the minority product of male-biasing conditions may show more

sents one of the most striking examples of phenotypic plasticity in male-typical morphology, physiology and/or behavior (Rhen and

that a single genotype can produce either male or female phen- Crews, 2002). Conversely, males produced as the minority product

otypes depending on the environmental conditions experienced of female-biasing conditions may exhibit a greater preponderance

during development (Kraak and Pen, 2002). Among vertebrates, of female-typical characteristics. These subtle effects of the sex

ESD has been observed in both ﬁsh and reptiles (Janzen and determination and differentiation process may be an important

Paukstis, 1991; Devlin and Nagahama, 2002). In reptiles, ESD is source of phenotypic variation (Crews and Groothuis, 2005).

Pelvicachromis pulcher is a species of small cichlid ﬁsh found in

rivers and streams in West Africa (Heiligenberg, 1965; Nwadiaro,

1985). P. pulcher are biparental substrate spawners which exhibit

∗

Corresponding author. Present address: Department of Psychology, Neuro-

long periods of parental care (Nelson and Elwood, 1997). P.

science and Behaviour, McMaster University, 1280 Main Street West, Hamilton,

pulcher males exist in two morphs deﬁned by the color of

Ontario, Canada L8S4L8. Tel.: +1 905 525 9140x26037; fax: +1 905 529 6225.

their opercula (Heiligenberg, 1965; Fig. S1 in the supplementary

E-mail addresses: [email protected], [email protected]

(A.R. Reddon). online, Appendix). Yellow opercula males (YO) are monogamous,

140 A.R. Reddon, P.L. Hurd / Zoology 116 (2013) 139–143

engaging in a single reproductive bout with a single female at any with several artiﬁcial plants, ﬂowerpots and lengths of black PVC

given time. Red opercula males (RO) are facultatively polygynous pipe to serve as potential nest sites. Fish were fed daily on dried and

and may breed monogamously or hold a harem of two or more frozen prepared cichlid foods and water temperature was main-

◦

females simultaneously, each of which maintain their own sub- tained at 26 ± 2 C for the duration of the study. Water in the stock

territories within the RO’s territory (Martin and Taborsky, 1997; aquaria and in the breeding aquaria before and after our manipula-

Barlow, 2000). YO males may also act as satellites on polygynous tions was held at pH 6.0 ± 0.1. These pH and temperature conditions

RO males’ territories (Martin and Taborsky, 1997). P. pulcher also mimicked the natural habitat of this species (Nwadiaro, 1985).

show a conspicuous sexual dimorphism at sexual maturity with the After 48 h acclimation time in the breeding tanks, we began the

females being shorter and deeper bodied with brighter coloration pH treatments. We randomly selected 4 of the 8 breeding aquaria

than the males which are more streamlined and less colorful (Fig. S2 and gradually lowered the pH to 5.5 ± 0.1 and in other 4 breeding

in the supplementary online, Appendix). Female coloration varies aquaria we gradually raised the pH to 6.5 ± 0.1. We based these

continuously between individuals, but females do not show distinct treatments on the previous study reporting pH-dependent ESD in

morphs. It has been assumed that the male morphs are genetically P. pulcher (Rubin, 1985) and on the range of pH values measured in

determined and ﬁxed throughout life (Heiligenberg, 1965), though their native habitat (Nwadiaro, 1985). We chose to manipulate pH

evidence for this claim is scarce. We know of no data as to the rel- prior to spawning in order to completely standardize the exposure

ative frequency of the RO and YO morphs in the wild, though both of the offspring to their pH conditions and ensure offspring were

subtypes are present (Martin and Taborsky, 1997). exposed to their treatments as soon as the eggs were laid. We main-

An earlier paper reported that P. pulcher show ESD dependent on tained our experimental pH using commercially available aquarium

water pH during development, with more acidic conditions produc- pH buffer (Seachem Laboratories, Madison, GA, USA) and we mon-

ing a greater proportion of males than do more neutral conditions itored the pH in our experimental aquaria daily using a portable

(Rubin, 1985). As with other instances of ESD in ﬁshes (e.g., Römer electronic pH meter (pHep 4; Hanna Instruments, Woonsocket, RI,

and Beisenherz, 1996), the environmental inﬂuence on sex is not USA).

absolute in P. pulcher and substantial numbers of the opposite sex The parent ﬁsh lived and bred within their experimental pH con-

are produced in sex-biasing pH conditions, allowing us to compare ditions. We conducted weekly water changes of 10 l using water

males and females from each developmental condition. that had been pre-buffered to match the pH treatment condition.

The goals of the current study were threefold. First, we wanted We checked the breeding aquaria daily for the presence of eggs

to replicate a previous study (Rubin, 1985) and conﬁrm that sex and all pairs spawned within 21 days of the onset of treatment.

is indeed inﬂuenced by developmental pH in P. pulcher. Second, We maintained the pH treatments for 30 days after the pair had

we wanted to determine whether pH conditions inﬂuenced the spawned, after which we allowed the aquaria to gradually return

expression of alternative male morphs, speciﬁcally, the ratio of to the standard pH (6.0) maintained in our laboratory. We allowed

RO to YO males, in an effort to determine whether male morph the parents to remain with their offspring and provide parental care

is genetically determined as previously suggested, or if this aspect until 60 days post-hatching, at which point we removed the parents

of the P. pulcher phenotype is also plastic and inﬂuenced by the and returned them to the stock aquaria. Following the removal of

same developmental conditions that inﬂuence sex. If RO males the parents, we split each clutch into two separate 110 l aquaria to

represent a more masculinized male form, it is conceivable that reduce density and speed maturation. Mortality was monitored and

male-producing pH conditions will also produce a higher propor- was rare. The vast majority of the F1 generation survived into adult-

tion of the RO type than do female-biasing pH conditions. Finally, hood, and there was no indication of differential mortality between

we wanted to determine whether developmental pH affected indi- the treatment groups.

vidual differences in behavior within each sex. If males are more

aggressive than females, then females that develop in male-biasing

2.2. Sexing

conditions may be more aggressive than females that develop in

female-biasing conditions (Rhen and Crews, 2002). Conversely,

We allowed the fry ∼330 days to mature into adults, at which

males that are the minority product of female-biasing pH may show

point we sexed all ﬁsh by visual inspection.

less male-typical aggression than do males from male-biasing con-

ditions. We predicted that acidic, male-biasing conditions would

2.3. Mass measurement

produce more males, more RO males and more aggressive individ-

uals of both sexes than would the more neutral, female-biasing pH

At ∼360 days post-hatching we weighed a sample of 160 ﬁsh (56

conditions.

males and 104 females, drawn equally from the two pH treatments)

to ±0.01 g using an electronic balance.

2. Materials and methods

2.4. Aggression testing

2.1. Breeding and pH treatment

At ∼400 days post-hatching, we tested 67 individuals (35 from

pH 5.5 and 32 from pH 6.5 including approximately equal numbers

We acquired a total of 40 P. pulcher from multiple local suppliers.

of each sex from each family) for aggressive behavior. The aggres-

We housed the P. pulcher together in two large communal aquaria

sive behavior test was conducted in a 19 l (25 cm × 25 cm × 30 cm)

(210 l, 120 cm × 32 cm × 52 cm). These ﬁsh served as parental stock

aquarium. Along one of the walls of the test aquarium was a mirror

for our breeding experiments. We selected 8 breeding pairs from

hidden behind a removable opaque Plexiglas partition. We placed

this initial stock. Parents were sexually mature and paired such

one ﬁsh into the aquarium and allowed it to acclimate and establish

that the male was larger than the female, mimicking the natural

a territory for 24 h. Following the acclimation period, we raised the

within-pair sexual size dimorphism. All breeding males appeared to

barrier hiding the mirror and allowed the ﬁsh to interact with its

be of the YO morph, though the breeders were not euthanized and

mirror image for 600 s. Trials were videotaped, and later scored for

thus parental morph was not conﬁrmed through the paraformalde-

aggression by a trained observer who was blind to the treatment

hyde technique (see Section 2.5). Each pair was placed into an

conditions. We counted the number of bites the ﬁsh delivered to

110 l (75 cm × 32 cm × 48 cm) breeding aquarium. Breeding aquaria

the mirror during the trial as a measure of aggression.

were furnished with a 3 cm layer of course sand as substrate along

A.R. Reddon, P.L. Hurd / Zoology 116 (2013) 139–143 141

2.5. Scoring of male morphs

Following aggression testing, the 67 behavior-tested individuals

were euthanized by immersing them in 0.2% 2-phenoxiethanol in

order to collect their brains for another study. Once all gill move-

ments ceased, we decapitated the ﬁsh and submerged their heads

in 25 ml of 4% phosphate buffered paraformaldehyde. Immer-

sion in paraformaldehyde immediately revealed the coloration in

the opercula that deﬁnes the RO and YO morphs (Fig. S3 in the

supplementary online, Appendix). This procedure allowed us to

x Ratio

unambiguously assign males to reproductive morphs, a process

that in live animals is complicated by the fact that the P. pulcher

male morphs are only discriminable when the ﬁsh are in breeding

condition (Heiligenberg, 1965) and that males immediately obscure

their opercular coloration when disturbed, rendering their opercula

a drab gray color for minutes to hours.

2.6. Statistical analysis

11 23 50 23 33 9 52 27

0.0 0.2 0.4 0.6 0.8 1.0

All statistical analyses were conducted using R (v 2.15.1). Mixed pH 5.5 pH 6.5

model analyses, when including clutch as a random effect, were

conducted using the lme4 library (v 0.999999-0). When the out- Fig. 1. Sex ratio plotted as the proportion of males for 8 broods that were raised

in two different pH conditions. Open bars = pH 5.5; shaded bars = pH 6.5. The total

come measure was continuous (i.e., mass of the individual), p values

number of ﬁsh in each brood is inset within each bar.

for the ﬁxed effects were calculated using MCMC techniques with

the function pvals.fnc() from the languageR library (v 1.4). MCMC

towards ﬁsh from lower (male-biasing) pH treatments to deliver

techniques cannot be used on glm mixed models in lme4, and so

more bites (Table 1, Fig. 2).

when the outcome was binary (i.e., sex of the individual) we used

Of the random sample of males’ heads that were ﬁxed in

the mixed model logistic regression, glmer() from lme4 and report

2 paraformaldehyde, a greater proportion of males from the pH 5.5

the test statistic it supplies to calculate p values for the ﬁxed

treatment were of the red opercula phenotype (13 of 18, compared

effects. We also applied the Anova() function from the compan-

to 2 of 15 in the pH 6.5 treatment; log-likelihood ratio: G1 = 7.2613,

ion to applied regression library (v2.0–12) to the glmer models.

p = 0.007, Fig. 3).

The Anova() function from the car library was also used when ana-

lyzing the bites per minute ANOVA in order to avoid sequentially

4. Discussion

removing the effects of the two independent variables, which is

the default for the native aov() function. Default arguments were

Consistent with a previous report (Rubin, 1985), we found that

used for all these functions, unless speciﬁed otherwise (e.g., selec-

sex determination is inﬂuenced by developmental pH in the West-

tion of the logistic regression for glmer() analysis when sex was the

African cichlid, P. pulcher. Although more females than males are

dependent variable).

produced at both pH 5.5 and pH 6.5, more males are produced at

pH 5.5 than are produced at pH 6.5. We also found that the male-

2.7. Ethical note

biasing treatment (pH 5.5) produced a higher proportion of the RO

male morph than did the female biasing treatment (pH 6.5). Finally,

Protocols were approved by the University of Alberta Biological

we found a trend towards heightened aggression in both males and

Sciences Animal Policy and Welfare Committee (protocol number

females from the pH 5.5 conditions compared to pH 6.5.

5441006) and adhere to the guidelines of the Canadian Council for

The natural habitat of P. pulcher ranges in pH from ∼5.5 to

Animal Care and the Animal Behavior Society.

∼

7, depending on site and season (Nwadiaro, 1985). The rainy

season may alter the pH of the rivers and streams which P. pul-

3. Results

cher naturally inhabit (Lowe-McConnell, 1991). P. pulcher breed

year round (Lowe-McConnell, 1991), and therefore ﬁsh born dur-

The sex ratios at ∼330 days of age for each of the eight clutches

ing the rainy season ought to have different sex and male morph

are plotted in Fig. 1. There was a signiﬁcant inﬂuence of pH on

ratios than ﬁsh born during the dry season. Fish born at different

sex ratio (logistic regression GLM, using glmer() with sex as out-

times of the year may face different challenges, and the relative ﬁt-

come variable, and clutch as random effect nested within the ﬁxed

ness of RO males, YO males and females may depend strongly on

effect of pH treatment; coded as: pH 5.5 = 0, pH 6.5 = 1; slope esti-

mate = −0.9044, slope standard error = 0.4567, z = −1.965, p = 0.049,

2 Table 1

= .

pH effect 1 3 92, p = 0.048). Clutches from the pH 5.5 treatment

Bites per minute as a function of sex and pH treatment. Note that clutch was not

produced more males than clutches from the pH 6.5 treatment

included as a variable in these analyses due to the small, and approximately equal,

although the overall sex ratio across treatments was female-biased

contribution of each clutch to the sample. Each clutch contributed between 2 and 6

(consistent with some natural populations; Nwadiaro, 1985). (mean = 4.1, sd = 1.4) females, and between 1 and 8 (mean = 4.0, sd = 2.6) males to the

A two-way mixed model with sex, pH and their interaction sample. Dropping the interaction term had negligible effect on the results (sex effect

p = 0.01, pH effect p = 0.11). Here we present the full analysis with the interaction

as ﬁxed effects, and clutch nested within both showed no effect

term as was planned a priori.

of pH manipulation on mass at ∼400 days post-hatching (pH

coded as above; estimated slope = 0.05, SE = 0.22, p = 0.18), although SS DF F P

males were heavier than females (females = 0, males = 1; estimated

Sex 350.51 1 6.81 0.01

slope = 1.8, se = 0.17, p < 0.001). pH 131.60 1 2.56 0.11

Sex × pH 34.15 1 0.66 0.42

Females delivered signiﬁcantly more bites per minute to their

Error 3138.17 61

mirror images than did males, and there was a non-signiﬁcant trend

142 A.R. Reddon, P.L. Hurd / Zoology 116 (2013) 139–143

color morphs are at least semi-permanent, consistent with previ-

ous reports that characterized these male types as a permanent,

lifelong dimorphism (Heiligenberg, 1965). Our results do, however,

contradict the assertion that male morph is entirely genetically

determined in P. pulcher. We found a strong inﬂuence of devel-

opmental pH on the ratio of RO to YO males that were produced.

ute Acidic male-biasing pH conditions produced predominantly RO

males, while more neutral, female-biasing pH conditions produced

predominantly YO males.

We found that pH during early development may also affect

10 15 20

the aggressiveness of the offspring produced. This result is remi-

Bites per mi

niscent of what has been found in the leopard gecko (Eublepharis

macularius) where high or low incubation temperatures produce a

preponderance of females, while intermediate temperatures lead

to an overproduction of males (reviewed in Crews and Groothuis,

2005). Interestingly, female geckos produced as the minority

product of male-biasing temperatures tend to behave in a male- 17 16 17 15

typical manner, acting more aggressively and in a more sexually

indiscriminant manner than females produced at female-biasing

Female Male

pH 5.5 pH 6.5 pH 5.5 pH 6.5 temperatures (Rhen and Crews, 2002; Crews and Groothuis, 2005).

Contrary to these patterns and to our predictions, female and not

Fig. 2. The number of bites per min delivered by ﬁsh to their mirror images. Females male P. pulcher were more aggressive in our assay. Our results,

delivered more bites than males (p = 0.01) but the apparent increase in ﬁsh from the

though non-signiﬁcant, do suggest that individuals that developed

pH 5.5 treatment was not signiﬁcant (p = 0.11), nor was the interaction between sex

in male-biasing conditions may be more aggressive than individ-

and pH (p = 0.42). Sample sizes are inset within each box.

uals that developed in female-biasing conditions. This is somewhat

paradoxical, considering that females appear to be the more aggres-

developmental conditions. Previously, it has been suggested that sive sex. It is possible that the masculinizing inﬂuence of the acidic

YO males may outperform RO males in the dry season while the pH condition does increase aggressiveness in both sexes, but that

converse is true during the rainy season (Martin and Taborsky, females are more aggressive than males overall due to some other

1997). mechanism unrelated to sexual differentiation.

One of the novel contributions of our study is that not only We cannot speak to the precise timing at which our manipu-

the sex ratio of P. pulcher, but also the ratio of polygynous RO lation has its effect, except to say that the sex-ratio modulation

males to monogamous YO males is affected by developmental occurs at some point between fertilization and 30 days post hatch-

pH. We serendipitously discovered that post-mortem immersion ing, which is consistent with previous work on ESD in ﬁshes (Römer

in paraformaldehyde allows for the unambiguous assignment of and Beisenherz, 1996). The fact that substantial numbers of both

P. pulcher males to their color morphs, a process that previously sexes were produced in both of our pH treatments suggests that

required extensive behavioral observations (Martin and Taborsky, pH is not the sole determinant of sex and that there is an additional

1997). Exposure to paraformaldehyde reveals the red or yellow col- effect of genetics and/or another environmental variable that also

oration in the opercula of male P. pulcher, which suggests that these plays a role in determining the sex of developing P. pulcher. This

ﬁnding is consistent with other ﬁsh species for which environ-

mental conditions have an effect on sex determination but do not

result in unisex clutches (Conover and Kynard, 1981; Römer and

Beisenherz, 1996). Sex determination in P. pulcher appears to be

environmentally inﬂuenced, but not environmentally determined.

We do not yet know the speciﬁc mechanism that ties sex ratio,

male morph, and perhaps aggressiveness to developmental pH O')

conditions in P. pulcher. A recent study on European sea bass

(Dicentrarchus labrax) found that increased temperatures during tion '

development increased the methylation of the gonadal aromatase

promoter gene (cyp19a; Navarro-Martín et al., 2011). This increased

methylation decreases expression of the aromatase enzyme, which

ph (propo

converts androgens to estrogens in the brain. As a result, higher

temperatures have a male-biasing effect on the sex ratio in Euro-

pean sea bass, which is mediated by the effect of temperature on

Male mo methylation of the aromatase promoter. It is conceivable that pH

may have a similar effect on some element of the aromatase sys-

tem resulting in a similar process occurring in developing P. pulcher,

with reduced pH decreasing the expression of aromatase resulting

in greater production of males. Aromatase could also be a prox-

18 15

imate determinant of the two alternative male phenotypes in P.

0.0 0.2 0.4 0.6 0.8 1.0

pulcher if the expression of the RO phenotype depends on reduced

pH 5.5 pH 6.5

expression of aromatase.

In conclusion, we conﬁrmed that sex determination is inﬂu-

Fig. 3. The proportion of males that were of the red opercula morph differed sig-

enced by developmental pH in the West African cichlid ﬁsh, P.

niﬁcantly between the two pH treatments (p = 0.007). The pH 5.5 condition, which

pulcher. Furthermore, we present evidence that the environmental

produced a higher proportion of males, also produced a higher proportion of males

that were of the red opercula morph. Sample sizes are inset within each bar. inﬂuence on phenotype extends to phenotypic expression within

A.R. Reddon, P.L. Hurd / Zoology 116 (2013) 139–143 143

each sex, altering the ratio of two alternative male phenotypes and Dingemanse, N.J., Kazem, A.J.N., Réale, D., Wright, J., 2009. Behavioural reaction

norms: animal personality meets individual plasticity. Trends Ecol. Evol. 25,

perhaps altering individual propensity for aggression within each

81–89.

sex. These ﬁndings suggest that the process of sexual differentiation

Heiligenberg, W.F., 1965. Colour polymorphism in the males of an African cichlid

may have an important effect on individual differences in behav- ﬁsh. J. Zool. 146, 95–97.

Janzen, F.J., Paukstis, G.L., 1991. Environmental sex determination in rep-

ioral and reproductive phenotypes. Our results show that P. pulcher

tiles: ecology, evolution, and experimental design. Quart. Rev. Biol. 66,

are a promising study system for testing adaptive hypotheses about 149–179.

environmental sex determination, alternative male reproductive Kraak, S.B.M., Pen, I., 2002. Sex determining mechanisms in vertebrates. In: Hardy,

I.C.W. (Ed.), Sex Ratios: Concepts and Research Methods. Cambridge University

tactics and the maintenance of individual variation in behavior.

Press, Cambridge, pp. 158–177.

Lowe-McConnell, R.H., 1991. Ecology of cichlids in South American and African

Acknowledgements waters excluding the African Great Lakes. In: Keenleyside, M.H.A. (Ed.), Cich-

lid Fishes: Behaviour, Ecology and Evolution. Chapman and Hall, London, pp.

60–85.

We thank Isaac Lank for constructing the aggression testing

Martin, E., Taborsky, M., 1997. Alternative male mating tactics in a cichlid, Pelvi-

apparatus, Cristian Gutiérrez-Ibánez˜ for his assistance in the lab cachromis pulcher: a comparison of reproductive effort and success. Behav. Ecol.

and Doug Wylie for providing research materials. This research was Sociobiol. 41, 311–319.

Navarro-Martín, L., Vinas,˜ J., Ribas, L., Díaz, N., Gutiérrez, A., Di Croce, L., Piferrer, F.,

supported by a Natural Sciences and Engineering Research Council

2011. DNA methylation of the gonadal aromatase (cyp19a) promoter is involved

of Canada (NSERC) Discovery Grant awarded to PLH. ARR is sup-

in temperature-dependent sex ratio shifts in the European sea bass. PLoS Genet.

ported by the Margo Wilson and Martin Daly Ontario Graduate 7, e1002447.

Scholarship. Nelson, C.T.J., Elwood, R.W., 1997. Parental state and offspring recognition

in the biparental cichlid ﬁsh Pelvicachromis pulcher. Anim. Behav. 54,

803–809.

Appendix A. Supplementary data Nwadiaro, C.S., 1985. The distribution and food habits of the dwarf African cich-

lid, Pelvicachromis pulcher in the River Sombreiro, Nigeria. Hydrobiologia 121,

157–164.

Supplementary data associated with this article can be found, in

Pigliucci, M., 1996. How organisms respond to environmental changes: from phen-

the online version, at http://dx.doi.org/10.1016/j.zool.2012.11.001. otypes to molecules (and vice versa). Trends Ecol. Evol. 11, 168–173.

Pigliucci, M., 2001. Phenotypic Plasticity: Beyond Nature and Nurture. Johns Hopkins

University Press, Baltimore, MD.

References

Pigliucci, M., 2005. Evolution of phenotypic plasticity: where are we going now?

Trends Ecol. Evol. 20, 481–486.

Barlow, G.W., 2000. The Cichlid Fishes: Nature’s Grand Experiment in Evolution. Rhen, T., Crews, D., 2002. Variation in reproductive behaviour within a

Perseus, Cambridge, MA, USA. sex: neural systems and endocrine activation. J. Neuroendocrinol. 14,

Bull, J.J., Vogt, R.C., 1979. Temperature-dependent sex determination in turtles. Sci- 517–531.

ence 206, 1186–1188. Römer, U., Beisenherz, W., 1996. Environmental determination of sex in Apistogram-

Conover, D.O., Kynard, B.E., 1981. Environmental sex determination: interaction of mai (Cichlidae) and two other freshwater ﬁshes (Teleostei). J. Fish Biol. 48,

temperature and genotype in a ﬁsh. Science 213, 577–579. 714–725.

Crews, D., Groothuis, A.G.G., 2005. Tinbergen’s fourth question: ontogeny. Anim. Rubin, D.A., 1985. Effect of pH on sex ratio in cichlids and a poecilliid (Teleostei).

Biol. 55, 343–370. Copeia 1985, 233–235.

Crews, D., Sakata, J., Rhen, T., 1998. Developmental effects on intersexual and Schlichting, C.D., 1986. The evolution of phenotypic plasticity in plants. Ann. Rev.

intrasexual variation in growth and reproduction in a lizard with temperature- Ecol. Syst. 17, 667–693.

dependent sex determination. Comp. Biochem. Physiol. C 119, 229–241. Thompson, J.D., 1991. Phenotypic plasticity as a component of evolutionary change.

Devlin, R.H., Nagahama, Y., 2002. Sex determination and differentiation in ﬁsh: an Trends Ecol. Evol. 6, 246–249.

overview of genetic, physiological, and environmental inﬂuences. Aquaculture West-Eberhard, M.J., 1989. Phenotypic plasticity and the origins of diversity. Ann.

208, 191–364. Rev. Ecol. Syst. 20, 249–278.