Journal of Fish Biology (1996) 48, 714–725

Environmental determination of sex in Apistogramma(Cichlidae) and two other freshwater fishes (Teleostei)

U. R̈ W. B

University of Bielefeld, Department of Biology, Postfach 100131, 33501 Bielefeld,Germany

(Received 15 July 1994, Accepted 30 June 1995)

Environmental sex determination by temperature could be revealed significantly in 33Apistogramma-species and in Poecilia melanogaster. In some, but not all, Apistogramma-speciespH also influences the sex ratio, whereas neither temperature nor pH affect the sex ratio ofPseudocrenilabrus multicolor victoriae. The sex in offspring of A. trifasciata is determinedwithin a sensitive period of about 30 to at least 40 days after spawning.

? 1996 The Fisheries Society of the British Isles

Key words: Apistogramma; Cichlidae; environmental sex determination; pH; sex ratio;temperature.

INTRODUCTION

Sex is determined in reptiles by at least two different mechanisms, one genotypicand the other environmental (Bull, 1980). In fish a genetic mechanism for sexdetermination was proposed long ago by Aida (1921). Since then it has beenestablished for most species which have been examined (Price, 1984). Determi-nation of sex by environmental factors after conception seems to be rare.Environmental factors such as pH in some cichlids and a poecilid (Rubin, 1985),and temperature in Menidia menidia (L.) (Conover & Kynard, 1981), M.peninsula Goode & Bean (Middaugh & Hemmer, 1987) and Poeciliopsis lucidaMiller (Sullivan & Schultz, 1986; Schultz, 1993) have been shown to influence sexratio, too. Hints in the aquarium literature motivated our investigations of theinfluence of temperature and pH on sex ratio in Apistogramma (Teleostei,Cichlidae). The objective of this article is to present further data in regard toenvironmental sex determination (ESD) in fish as this seems to be more commonthan currently appreciated.

MATERIALS AND METHODS

Thirty-seven species of Apistogramma, a genus of neotropical cichlids, Pseudo-crenilabrus multicolor victoriae Seegers, an African mouth-breeding cichlid, and Poecilia(Limia) melanogaster (Günther), a Jamaican live-bearing poecilid (Table I), were used forthis study. Nomenclature of Apistogramma-species follows Ufermann et al. (1987). Allfish were of known geographical origin which might be of special importance for furtherecological investigation.Generally, specimens taken from the wild and their F1-progeny were used for the

experiments with Apistogramma-species. There was no significant difference in sex ratioof offspring in specimens from different imports or strains of long-time inbred laboratory-stocks (F12 up to F15). Pseudocrenilabrus m. victoriae were F1-progeny of specimens

714

0022–1112/96/040714+12 $18.00/0 ? 1996 The Fisheries Society of the British Isles

T

I.Meanpercentageofmaleswithinbroodsof39Teleostei

Species

23)C

26)C

29)C

pH4·5

pH5·5

pH6·5

pH4·5

pH5·5

pH6·5

pH4·5

pH5·5

pH6·5

Apistogramma

agassizii(Steindachner)

42·2

(1)

77·3

(7)

—60·2

(1)

——

90·1

(1)

73·0

(6)

—borellii(Regan)

39·6

(1)

37·1

(3)

32·8

(1)

68·4

(2)

53·9

(2)

68·4

(2)

85·4

(1)

73·6

(4)

61·4

(1)

cacatuoidesHoedeman

—19·8

(5)

—84·3

(5)

62·7

(5)

43·3

(5)

—83·0

(5)

—caeteiKullander

59·9

(5)

53·3(12)

3·8(1)

43·7

(3)

48·0(14)

14·5

(8)

37·5

(3)

52·0

(9)

4·4(10)

diplotaeniaKullander

55·4

(1)

43·9

(3)

——

53·0

(2)

—98·8

(1)

79·9

(4)

—eunotusKullander

44·7

(2)

38·5

(2)

31·1

(2)

63·4

(1)

53·4

(3)

41·2

(2)

76·6

(2)

64·1

(2)

66·8

(2)

geisleriMeinken

—28·7

(2)

——

50·9

(3)

——

82·4

(2)

—gephyraKullander

45·8

(3)

40·6

(3)

35·5

(3)

65·7

(3)

56·1

(3)

39·2

(3)

78·0

(3)

62·4

(3)

66·8

(3)

gibbicepsMeinken

—19·2

(3)

—68·9

(2)

53·6

(2)

35·1

(2)

92·6

(2)

82·0

(2)

76·0

(2)

gosseiKullander

—28·7

(2)

——

49·2

(2)

——

81·4

(2)

—hippolytaeKullander

37·7

(1)

53·3

(2)

——

49·4

(1)

—93·2

(1)

85·7

(2)

—hoigneiMeinken

—30·9

(3)

——

——

—81·7

(4)

—hongsloiKullander

35·4

(3)

29·3

(3)

23·6

(3)

61·6

(3)

27·5

(3)

14·4

(3)

92·8

(3)

79·6

(3)

81·5

(4)

inconspicuaKullander

38·9

(2)

27·2

(2)

16·7

(2)

——

——

76·8

(2)

—iniridaeKullander

—34·4

(3)

——

——

—79·6

(2)

—linkeiKoslowski

—15·9(17)

—66·2

(7)

49·2

(7)

34·2

(6)

—88·9(13)

—luelingiKullander

—30·0

(2)

——

——

—88·5

(2)

—macmasteriKullander

—30·4

(2)

——

50·9

(2)

——

86·7

(2)

—meinkeniKullander

47·5

(1)

37·6

(2)

40·4

(1)

60·3

(1)

55·4

(2)

45·9

(1)

68·7

(1)

65·2

(2)

60·9

(1)

T

I.Continued

Species

23)C

26)C

29)C

pH4·5

pH5·5

pH6·5

pH4·5

pH5·5

pH6·5

pH4·5

pH5·5

pH6·5

Apistogramma

mendeziRöm

er—

35·8

(2)

——

——

—87·2

(2)

—nijsseniKullander

14·4(14)

9·9(12)

7·5(13)

58·7(20)

49·6(24)

45·9(21)

94·8(12)

85·0(18)

75·5(16)

norbertiStaeck

31·9

(2)

27·3

(2)

15·7

(2)

71·4

(2)

53·3

(2)

31·1

(2)

—89·2

(3)

—ortmanni(Eigenmann)

—25·0

(2)

——

48·8

(1)

——

82·9

(2)

—paucisquamisKullander&Staeck

43·2

(2)

—31·1

(2)

67·2

(1)

52·9

(1)

41·1

(1)

76·3

(2)

—67·0

(2)

pertensis(Haseman)

—29·3

(6)

——

——

—76·4

(6)

—resticulosaKullander

50·5

(2)

—25·7

(2)

——

38·7

(2)

90·3

(2)

—75·9

(2)

staeckiKoslowski

27·8

(2)

——

54·3

(2)

——

91·5

(2)

——

steindachneri(Regan)

41·1

(3)

—19·3

(3)

——

—90·9

(4)

—77·0

(2)

trifasciata(Eigenmann&Kennedy)

—16·8(15)

——

49·0(15)

——

85·9(15)

—uaupesiKullander

34·9

(3)

26·4

(3)

—81·7

(3)

58·8

(2)

—97·5

(3)

83·8

(2)

—‘Breitbinden’sp.*

—26·3

(5)

—79·2

(2)

68·5

(2)

——

89·3

(5)

65·0

(1)

‘Gelbwangen’sp.*

—31·1

(3)

——

——

—70·6

(2)

—‘Orangeschwanz’sp.*

—34·3

(3)

—61·1

(1)

—33·3

(1)

—67·2

(3)

—‘PuertoNarino’sp.*

—26·6

(3)

——

——

—79·6

(3)

—‘RioBranco’sp.*

—37·8

(3)

——

——

—67·4

(3)

—‘Rotpunkt’sp.*

38·4

(2)

25·9

(3)

——

50·9

(3)

36·0

(2)

84·9

(2)

77·3

(1)

62·2

(2)

‘Smaragd’sp.*

66·7

(2)

—62·6

(2)

76·2

(2)

75·9

(1)

61·5

(1)

75·6

(2)

—84·9

(2)

Poecilia

(Limia)

melanogasterSeegers

46(15)

41(15)

29(10)

59(12)

49(14)

38(25)

71(10)

67(18)

58(17)

Pseudocrenilabrus

m.victoriae(Günther)

48·3(29)

50·7(35)

50·3(29)

47·7(34)

53·9(43)

49·1(55)

51·6(19)

46·4(12)

52·5(17)

Figuresinparenthesesindicatenumberofbroods.

*Undescribedspecies.NamesusedinGerman

aquarium

literature.

caught in the wild by the subspecies-describer. Poecilia melanogaster used in ourinvestigations were descended from a laboratory-stock which was bred for about 25generations in captivity.All fish were bred in the laboratory at constant water conditions. Temperature, pH,

and water hardness were measured electronically. Deviation in temperature was less than&0·2) C, deviation of pH less than &0·5. Water was purified by filtration and by weeklychanges of 30% of water. If water was changed it was brought to the experimentalconditions in advance. Manipulation of pH was established by humic acid, sodiumbiphosphate and sodium bicarbonate.For the experiments, pairs as well as groups of five males and 25 females were bred in

150#50#40 cm tanks. No statistical differences of sex ratio were found between singlepairs and groups, for instance neither between 15 different pairs of Apistogramma nijsseniKullander nor between four different groups of A. linkei Koslowski. Spawning inartificial caves (46#30 mm, transparent boxes) was observed continually during thespawning period of about 48 h in temperature reversal experiments or at least daily instandard experiments by one of the authors (UR). The spawning period was regulated byfeeding the animals ad libitum with nauplii of brine shrimps (Artemia sp.). The numbersof eggs, larvae or fry were counted regularly. Females and clutches were transferred fromthe spawning tank to rearing tanks after fertilization. Females were removed from theirbrood after the offspring became free swimming, which was usually after 7 to 10 daysfrom hatching.Fish were bred and raised at 23, 26 and 29) C, and pH 4·5, 5·5 and 6·5, respectively.

Though different pH-schemes could not be investigated in all cases, we present all ourdata of environmental sex determination (ESD) by pH, as there is strong evidence thatthe sex of a variety of Apistogramma-species is influenced both by temperature and pH(Table I). In general, clutches were bred at pH 5·5 as losses of eggs, larvae or fry werelowest at this pH. Our investigations of the influence of temperature were focused mainlyon A. borellii (Regan), A. nijsseni, A. trifasciata (Eigenmann & Kennedy), and A. caeteiKullander. To determine at what stage of development ESD took place, eggs, larvae, andfry were transferred at different times from 23 to 29) C, and the reverse. The moment thelast egg of a clutch was fertilized we chose as time zero. The maximum duration of aspawning we observed was 1 h, therefore differences in age of eggs within a clutch (error)do not exceed this time in our experiments. Sex was determined according to sexuallydimorphic characteristics which were described by Kullander (1980, 1986).For statistical purposes, experiments were used only if the total loss of eggs and

offspring within a brood was less than 10%. Therefore, differential mortality did notsignificantly affect the results. The CSS-Statistica program was used for statisticalcalculations. Data for all temperature/pH-combinations are not yet available for allspecies, and only the data of seven Apistogramma-species were examined by multivariatecorrelation analysis (two-way ANOVA) which can handle unbalanced data (Table II). Inaddition, the correlation between temperature, pH, and sex ratio for all species examined

T II. Statistical significance of correlation from multivariatecorrelation analyses (two-way ANOVA) of sex ratio and temper-ature (T ), pH, and temperature#pH in Apistogramma-species

Species P (T ) P (pH) P (T#pH) ÷2 d.f.

A. borellii 0·000924 0·055775 0·791684 2·690 3A. caetei 0·496003 0·000000 0·303825 15·282 6A. eunotus 0·000001 0·000796 0·168134 2·566 2A. gephyra 0·000000 0·000001 0·036653 9·665 4A. hongsloi 0·000000 0·000000 0·000002 15·103 4A. meinkeni 0·004540 0·061353 0·361128 2·884 1A. nijsseni 0·000000 0·000000 0·003309 75·714 8

717

T III. Spearman rank correlation for temperature and pH

SpeciesOffspring/brood

R (T ) P (T ) R (pH) P (pH)mean min max n

Apistogrammaagassizii 85·6 51 133 16 0·89 <0·001 0·00 1·0borellii 72·2 25 122 17 0·79 <0·001 "0·52 0·14cacatuoides 68·5 34 97 25 0·60 <0·001 "0·72 <0·001caetei 77·8 19 174 65 "0·35 <0·004 "0·11 <0·37diplotaenia 59·8 31 81 11 0·89 <0·001 "0·29 0·37eunotus 113·4 56 155 18 0·87 <0·001 "0·37 0·12geisleri 62·0 33 87 7 0·94 0·001 — —gephyra 88·6 43 143 27 0·76 <0·001 "0·50 <0·008gibbiceps 71·3 42 109 15 0·93 <0·001 "0·33 <0·22gossei 62·3 49 73 6 0·95 <0·001 — —hippolytae 82·7 62 114 7 0·84 <0·016 "0·15 0·73hoignei 55·9 42 69 7 0·86 <0·012 — —hongsloi 52·2 15 90 28 0·70 <0·001 "0·41 0·028inconspicua 40·5 24 58 8 0·86 <0·030 — —iniridae 77·2 70 83 5 0·86 <0·058 — —linkei 41·8 19 68 50 0·93 <0·001 "0·24 0·087luelingi 76·5 54 93 4 0·89 <0·106 — —macmasteri 90·2 73 117 6 0·95 <0·003 — —meinkeni 65·0 47 83 12 0·91 <0·001 "0·30 0·33mendezi 75·8 54 97 4 0·89 <0·106 — —nijsseni 58·5 24 121 150 0·92 <0·001 "0·19 0·015norberti 95·9 26 215 15 0·83 <0·001 "0·33 0·22ortmanni 48·4 41 57 5 0·94 <0·014 — —paucisquamis 74·6 59 116 11 0·87 <0·001 "0·36 0·27pertensis 37·4 19 59 12 0·86 <0·001 — —resticulosa 63·2 37 88 10 0·81 <0·004 "0·49 0·14staecki 51·8 31 67 6 0·95 <0·001 — —steindachneri 89·5 51 141 12 0·86 <0·001 "0·56 <0·057trifasciata 46·2 22 80 45 0·94 <0·001 — —uaupesi 48·3 29 80 16 0·89 <0·001 "0·42 0·10

‘ Breitbinden ’ sp.* 62·2 29 100 15 0·86 <0·001 "0·17 0·53‘ Gelbwangen ’ sp.* 59·4 35 78 5 0·86 <0·058 — —‘ Orangeschwanz ’ sp.* 153·9 111 191 8 0·88 <0·004 "0·21 0·60‘ Puerto Narino ’ sp.* 86·8 67 122 6 0·87 0·021 — —‘ Rio Branco ’ sp.* 68·7 55 88 6 0·87 0·021 — —‘ Rotpunkt ’ sp.* 79·9 36 151 15 0·88 <0·001 "0·14 0·60‘ Smaragd ’ sp.* 78·8 23 181 12 0·73 <0·001 "0·02 0·93Poecilia (Limia)melanogaster 28·4 12 45 136 0·79 0·011 "0·57 0·10

Pseudocrenilabrusm. victoriae 25·2 6 41 273 0·15 0·68 0·10 0·78

Offspring/brood: mean: mean of all broods of a species; min and max: minimal and maximal absolutenumber of offspring within a single brood; R (T ): Spearman rank correlation coefficient for temperature;R (pH): Spearman rank correlation coefficient for acidity; P (T ) and P (pH): level of significance.*Undescribed species. Names used in German aquarium literature.

718 . ̈ .

was tested using the Spearman rank correlation coefficient, especially designed for smallersamples of non-parametric data (Sachs, 1984).In most temperature schemes the influence of pH on the sex ratio of the offspring was

tested. For estimation of the temperature effect on sex ratio all data from differentpH-values within each temperature scheme were pooled before being tested usingSpearman’s method (Table III).All experiments have been carried out in accordance with the national law. Voucher

specimens are deposited at the Zoologisches Forschungsinstitut and Museum AlexanderKoenig, Bonn, Germany.

RESULTS

In Apistogramma borellii, A. eunotus Kullander, A. gephyra Kullander, A.hongsloi Kullander, A. meinkeni Kullander and A. nijsseni temperature was thesignificant dominating factor affecting sex ratio. Whereas in A. caetei there wasno statistically significant impact of temperature on the sex ratio which was,however, strongly influenced by pH (Table II; Fig. 1). Though not statisticallysignificant, there seemed to be a tendency in A. caetei to produce relatively higherrates of males at 23 than at 29) C, which would be the reverse of observations inother species.In the Apistogramma-species, for which data have been analysed by multi-

variate correlation analysis, some influence of pH on the sex ratio could be seen(Table II; Fig. 1). The lower levels of significance in A. borellii, A. eunotus andA. meinkeni may be a result of the relatively small data sets in these species.In total, the correlation between temperature and sex ratio of offspring of 33

species of Apistogramma was significant (Table III). For example, the sex ratioof offspring in A. trifasciata at 23) C was skewed towards females, at 29) Ctowards males, and at 26) C the number of males and females was approximatelybalanced (Fig. 2). Four other species of Apistogramma seemed to show the sameresponse to temperature, but significance could not be shown since only four orfive broods could be raised for this investigation. Poecilia melanogaster showedthe same response to temperature as most species of Apistogramma, at 23) C thesex ratio of offspring was skewed towards females, at 29) C towards males, andat 26) C the sex ratio was again approximately balanced, whereas neithertemperature nor pH influenced the sex ratio of offspring in Pseudocrenilabrus m.victoriae (Table III).To determine the stage of development at which temperature influenced the

sex ratio in Apistogramma, eggs, larvae or fry of A. trifasciata were transferred atdifferent developmental stages from a male-determining temperature (23) C) to afemale-determining temperature (29) C) [Fig. 3(a)], and the reverse [Fig. 3(b)].Both experiments revealed that the influence of temperature depends on the timeafter which the brood is transferred. Broods transferred 800 h or later from theend of spawning (time zero) had sex ratios characteristic of the initial tempera-ture. Those transferred within 0 to 72 h had sex ratios characteristic of the finaltemperature. Change in sex ratio was linear for broods transferred at laterintervals up to 800 h, indicating that temperature effects are cumulative duringthe first 800 h of development.According to Rubin (1985) pH affects the sex ratio in Apistogramma borellii

and A. caucatoides Hoedeman (which is probably a spelling mistake for

719

5.5

100 0

6.5

50

4.5

T ×

pH

5.5

100 0

6.5

50

4.5

5.5

100 0

6.5

50

4.5

5.5

100 0

6.5

50

4.5

5.5

100 0

6.5

50

4.5

5.5

100 0

6.5

50

4.5

5.5

100 0

6.5

50

4.5

100 050

pH

100 050

100 050

100 050

100 050

100 050

100 050

5.5

6.5

4.5

5.5

6.5

4.5

5.5

6.5

4.5

5.5

6.5

4.5

5.5

6.5

4.5

5.5

6.5

4.5

5.5

6.5

4.5

100 050

(a)

T

100 050

(b)

100 050

(c)

100 050

(d)

100 050

(e)

100 050

(f)

100 050

(g)

2629

2326

2923

2629

2326

2923

2629

2326

2923

2629

23

F.1.Percentageofmalesdependenton

temperature(T),top;pH

,centreandtemperatureandpH

(T#pH

)lowerinApistogramma-species.(a)A.borellii;

(b)A.caetei;(c)A.eunotus;(d)A.gephyra;(e)A.hongsloi;(f)A.meinkeni;(g)A.nijsseni.T

#pH

:1=29,,=26and

#=23

)C.

A. cacatuoides Hoedemann in Rubin’s article). Therefore we investigated alsothe influence of pH on a variety of Apistogramma-species. In some but not allApistogramma-species the sex ratio of offspring was biased significantly by pH(Tables I, III). The influence of pH is quite different from that of temperature.In A. nijsseni for instance the influence of temperature seemed to be thedominant factor [Fig. 4(a)], whereas in A. borellii both, high temperature and lowpH level, revealed a high percentage of males [Fig. 4(b)].

DISCUSSION

Whereas genotypic sex determination has been shown in many fish species(Price, 1984), ESD in fish has been shown so far in only a few (Conover &Kynard, 1981; Rubin, 1985; Sullivan & Schultz, 1986; Middaugh & Hemmer,1987; Schultz, 1993). Our experiments prove that sex is determined in most, ifnot all, Apistogramma-species and in Poecilia melanogaster by environmentalfactors such as temperature and pH, and ESD in fish may be more common thancurrently appreciated.In contrast to temperature-dependent ESD in reptiles which typically exhibit

predominately all male or all female sex ratios over a wide range of incubationtemperatures (e.g. Ferguson & Joanen, 1982; Standora & Spotila, 1985; Bullet al., 1990; Wibbels et al., 1991; Viets et al., 1993), in Apistogramma-species, asin Menidia menidia (Conover & Heins, 1987a, b), ESD by temperature is not an‘ all-or-none ’ effect. If incubation temperature during the first days afterfertilization is changed from 26) C, a temperature which yields a balanced sexratio, to a warmer (29) C) or cooler (23) C) temperature, respectively, sex ratiosin offspring of Apistogramma shift in both cases about the same amount, but inopposite directions. Thus, we cannot find differences in sex determining poten-cies with regard to the different temperatures we tested. Otherwise unbalancedchanges of sex ratios should be expected under these conditions, as Bull et al.(1990) found in turtles.

100

0

Temperature (°C)

Mal

e (%

)

80

60

40

20

23 26 29

F. 2. Influence of temperature on sex ratio in Apistogramma trifasciata. n=15 at each temperature.Mean; standard error ([ ]); standard deviation (T).

721

The sex of progeny of Apistogramma-species is therefore not determined atconception, but within a sensitive period extending from 0–35 days afterspawning. This agrees with findings that heteromorphic sex chromosomes areabsent in all neotropical cichlids (Kornfield, 1984).Sex determination in the Atlantic silverside (M. menidia) is under control of

both genotype and temperature during a specific period of larval development

1000

100

00

Time (h)

Mal

e (%

)

80

60

40

20

200 400 600 800 1400

Sensitive period

(b)

1000

100

00

Time (h)

Mal

e (%

)80

60

40

20

200 400 600 800 1400

Sensitive period

(a)

F. 3. (a) Percentage of males after transfer of eggs, larvae or fry of Apistogramma trifasciata at differenttimes from 23) C, at which temperature reveals a low percentage of males, to 29) C, at whichtemperature reveals a high percentage of males. (b) Percentage of males after transfer of eggs,larvae or fry of Apistogramma trifasciata at different times from 29) C, at which temperaturereveals a high percentage of males, to 23) C, at which temperature reveals a low percentage ofmales. Mean; standard error ([ ]); standard deviation (T); arrow: expected percentage of males ifclutch is not transferred to lower/higher temperature.

722 . ̈ .

100

6.5

pH-level

Mal

e (%

)

80

60

40

20

5.5

4.5 23

26

29

Temperature (°C)

100

6.5

pH-level

Mal

e (%

)

80

60

40

20

5.5

4.5 23

26

29

Temperature (°C)

(a)

(b)F. 4. (a) Influence of temperature and pH-level on sex ratio in Apistogramma nijsseni. (b) Influence of

temperature and pH-level on sex ratio in Apistogramma borellii.

723

(Conover & Kynard, 1981; Conover, 1984). The hypothesis of ESD suggestsenvironmental determination of sex whenever the environment affects therelative fitness of males and females differentially and when the environmentalconditions that offspring enter cannot be chosen (Charnov & Bull, 1977). Inaddition, parental adjustment of sex ratio in offspring during the period ofcare may affect the reproductive success of male and female offspring differ-ently (Trivers & Willard, 1973). Adaptive variation in environmental andgenetic sex determination in M. menidia according to this hypothesis could bedemonstrated by Conover & Heins (1987a, b): populations at different latitudescompensate for differences in thermal environments and seasonality by adjust-ing the response of sex ratio to temperature, and by altering the level ofenvironmental control in contrast to genetic control. Large size, as the resultof a longer growing season, enhances the survival and fecundity of femaleM. menidia, whereas males do not appear to be greatly affected by body size.Thus, it is an advantage to populations in the south with long breedingseasons to hatch females by ESD first, whereas populations in the north withshort breeding seasons do not.Typical Apistogramma are small, shelter-breeding fish, living in the leaf litter of

small rivers and brooks. They are in most cases polygamous, but in rarer casesmonogamous. Females care for their brood for about 10–50 days. Until now,there has been no demonstration of either any ecological significance of ESD orany selective evolutionary advantage for the occurrence of ESD inApistogramma-species. But recent investigations in brooks of the Rio Negrosystem (Brazil) by one of us (UR) revealed a temperature gradient with respectto water depth, ranging from about 30) C or more near the surface to 23) C orless at the bottom. No pH-gradient was found between different strata of thewater, but there was one at different times of the day. The temperature gradientwas correlated with different numbers of individuals and different percentages ofmales (Römer, unpubl.), while no such effect was found in relation to the pH.Thus, we presume that our findings of an ESD in the laboratory is of importancein the wild, too. Preliminary field studies seemed to reveal a socially influencedtemperature scheme for ESD in Apistogramma, instead of a seasonally orgeographically influenced one, as found inMenidia, but further investigations arenecessary.All investigated Apistogramma-species, except A. caetei, seemed to follow a

common pattern of control of the sex ratio, especially in regard to temperature(Fig. 1), whereas A. caetei seemed to show an inverse response of sex ratio totemperature and additionally, a high response to the pH-level. No reason forthis can be found in experimental design nor in available ecological data. Atpresent we can only speculate that differences in habitat distribution, com-pared to other Apistogramma-species investigated by us, may cause theeffect. A. caetei is found in streams and brooks in the vicinity of Belém whileall the other species originated from the Amazon, Orinoco or Paraguay Basins.A. caetei, as well as two additional Apistogramma-species which were notavailable until now, represent the most eastern forms of the genus from theBrazilian Shield. This may result in different ecological conditions anddiffering temperature and pH regimes, leading to an altered ESD system inA. caetei.

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