LABORATORY STUDIES BEARING ON PIGMENT ... - genetics.org · LABORATORY STUDIES BEARING ON PIGMENT PATTERN POLYMORPHISMS IN WILD POPULATIONS OF RANA PIPIENS DAVID J. MERRELL Department

LABORATORY STUDIES BEARING ON PIGMENT PATTERN POLYMORPHISMS IN WILD POPULATIONS OF

R A N A PIPIENS

DAVID J. MERRELL

Department of Zoology, University of Minnesota, Minneapolis, Minnesota 55455

Manuscript received December 8, 1970 Revised copy received June 11, 1971

ABSTRACT

Data are presented for 2,393 progeny from a number of crosses related to a study in ecological genetics of the Burnsi and Kandiyohi polymorphisms in natural populations of the leopard frog, R a m pipiens. No significant differences in viability were found between wild-type homozygotes (+/+) and Burnsi heterozygotes (B/+) or homozygotes ( B / B ) . Similarly, no difference in viability was found between wild-type (+/+) and Kandiyohi heterozygotes ( K / + ) and homozygotes ( K / K ) . However, there appears to be slight reduction in viability of the double dominant heterozygote (B/+; K/+) in comparison with (+/+), ( E / + ) , and (K /+) progeny from the same cross.--The Kandiyohi heterozygotes (K/+) appeared to have a more rapid rate of develop ment from fertilization through metamorphosis than wild-type (+/+) or Burnsi (B+/) or Burnsi-Kandiyohi heterozygotes ( B + K/+) . Since Kan- diyohi is associated primarily with the prairie habitat (MERRELL 1965), this finding suggests that the adaptive advantage of Kandiyohi lies in the more rapid rate of development of frogs carrying this gene, enabling them to complete metamorphosis before the prairie breeding ponds dry up.-Data are presented from crosses involving dorsal spot number. The results suggest that heredity plays a role in the determination of dorsal spot number but that non-genetic influences are also of considerable importance.-The results of these crosses are discussed with respect to their bearing on the formation of pigment patterns in Rana pipiens. From the available data it is clear that the pigment pattern in R a m pipiens is a complex trait influenced by major gene loci, by modifying genes, and by environmental effects. The relatiye importance of these factors varies depending on the particular combination of genetic and environmental conditions.

W E E D (1922) tentatively named two new species of frogs from Minnesota, Rana burnsi and Rana kandiyohi. MOORE (1942) showed that the unspotted

Burmi type results from a single dominant gene B inL,the leopard frog, Rana pipiens; VOLPE (1955) showed that the mottled Kandiyohi type results from a different dominant gene K , unlinked to B (VOLPE 1956; ANDERSON and VOLPE 1958; VOLPE 1960). MERRELL (1965) found naturally occurring polymorphism for the B and K genes to be frequent in populations in Minnesota and adjacent areas.

Genetic polymorphisms have been described in a variety of species, but under- Genetics 70: 141-161 January 1972.

142 DAVID J. MERRELL

standing of the adaptive significance of most of these polymorphisms is still limited. Efforts to study such problems have required a fusion of the concepts of population ecology and population genetics into an emerging field known as ecological genetics.

Ecological genetics is the study of the adaptation of natural populations to their physical and biological environments and of the mechanisms by which they re- spond to environmental change ( FORD 1964; MERRELL, in press). I t is, in essence, the direct study of the evolutionary process at the level at which evolution actually occurs. Studies in ecological genetics ordinarily require both field and laboratory investigations. Classic studies are those of CLAUSEN and HIESEY (1958) on ecological races in Potentilla, KETTLEWELL (1961 ) on industrial melanism in moths, and DOBZHANSKY (1970) on inversion polymorphism in Drosoph- ila. The results of some of the field work on the B and K polymorphisms have already been reported (MERRELL I965,1968,1969a, 1969b, 1970; MERRELL and RODELL 1968). This paper presents the results of laboratory studies bearing on the adaptive significance of these dominant pattern genes, in particular the data on 2393 progeny from a number of crosses. In addition, the work of others on pigment patterns in Rana pipiens is reviewed and discussed and an attempt is made to reconcile some of the differences that have emerged. In the course of these studies, data were collected on growth and the attainment of sexual maturity. This information is attached as Appendices A and B.

MATERIALS A N D METHODS

The methods used to make crosses and to rear the tadpoles through metamorphosis have already been described (MERRELL 1963). In brief, the eggs were fertilized by the methods out- lined by RUGH (1962), the egg masses transferred to large finger bowls until the embryos had developed into small swimming tadpoles (Shumway stage 24), at which time they were transferred to large floor tanks to complete development. The smallest tanks used were 7 ' ~ 3 1 / 2 ' ~ 2 i / ' . The untreated well water was kept a t about one foot in depth and was not changed. The primary food was boiled lettuce. In this way fairly large numbers of tadpoles were reared with a mini- mum of care.

The frogs used in the crosses were either captured in the vicinity of the Twin Cities or obtained from Schettle Biologicals, Stillwater, Minnesota. Recently captured wild frogs were used whenever possible because their physical condition was usually superior to that of frogs held in the dealer's tanks.

I. CROSSES INVOLVING T H E WMINANT GENES

A. RELATIVE VIABILITY OF MUTANT AND WILD PHENWI'YPES

Since the Burnsi and Kandiyohi genes occur at polymorphic frequencies in natural populations, it seemed desirable to test their viability under somewhat crowded conditions in the hope that this stress might help to reveal any differences in viability among the various possible phenotypes.

The evidence concerning crowding is mostly indirect. Counts have shown (MERRELL, 1965) that a single female produces from 2,000 to 5,000 eggs. The success of the crosses in terms of the percentage of eggs fertilized varied, apparently primarily in relation to the condition of the eggs. All of the surviving embryos from each cross were maintained, during the early stages in the finger bowls, and later in the floor tanks. The number of embryos per finger bowl was ordinarily of the order of at least 100, as compared to the generally recommended number of 25 or less,

P I G M E N T PATTERNS IN FROGS 143

and mortality was usually so high that it was necessary to remove the dead embryos almost daily. When the 7- to IO-day old tadpoles were transferred to the floor tanks, crowding was probably less severe. Nevertheless, metamorphosis, rather than being completed by all tadpoles in less than a week as is characteristic of natural populations, occurred over periods ranging from 2 to 6 months or more. This extended metamorphic period undoubtedly reflected the inhibitory effects of crowding on the development of the surviving tadpoles (RICHARDS 1958; ROSE 1960). Since the primary interest was in the relative survival of the different genotypes when reared together, no attempts were made to keep counts of the number of surviving tadpoles a t various stages. However, the greatest number of progeny surviving through metamorphosis for any one cross was 305, which represents a survival rate of somewhere between 6% (305/5000) and 15% (305/2000). In most of the crosses, therefore, survival must have been of the order of 5% or less. If crowding could enhance differences in viability, these conditions seem stringent enough to have brought out such differences.

RESULTS

The data in line 1 of Table 1 on 539 progeny from crosses of heterozygous Burnsi (I?/+) and wjld tyne (+/+> indicate no cienjficant difference in viability between B/+ and +/+ frogs. This finding is similar to that of other investigators, as shown iE Table 1, and even the summed data do not give a significant x2 value. There is certainly no indication of heterozygous advantage.

Table 2 gives the results from crosses of B / + x B / + frogs plus similar data from four other investigators. In one case with small numbers, the deficiency of the wild type is significant at the 5% level, but the overall data show no significant deviation from the expected 3 Burnsi to 1 wild type ratio. Just as the crosses in Table 1 show no heterozygous advantage for B / + , these results do not indicate any disadvantage for BIB homozygotes, a possibility suggested by the reduced fitness of dominant homozygotes for dominant polymorphic pattern genes in the grouse locust (NABOURS 1929; FISHER 1939). The results, therefore, give no indi- cat;on that either the B / B or the B/+ Burnsi frogs differed significantly from the wild type (+/+) in viability.

TABLE 1

Crosses between Burnsi heterozygotes (B/ f ) and wild type (+/+)

Progeny (N) Investigator +/+ B / + Xa

MERRELL 288 251 2.54 MOORE (1 942) B x + 108 111 0.04

+ X B 102 85 1.54 MOORE (1943) B x Vt. + 23 33 1.78

B x Okl. + 31 29 0.06

(1961) 32 37 0.36 (1970) 54 48 0.35

DAVISON (1961) + x B* 12 16 0.57

N A C E ~ ~ Q ~ . (1970) + x B 58 52 0.33 Total 766 716 1.68

VOLFE (1960) 40 36 0.21

B X + 18 18 0.00

* One spontaneous triploid omitted.


TABLE 2

Crosses between Burnsi heterozygotes (B/+ x B/+)

Investigator

MERRELL MOORE (1942)

VOLPE (1956) DAVISON (1961) NACE et al. (1970) Total

(1943)

Progeny (N) +/+ B / - X=

10 49 2.04 77 224 0.05 6 39 3.27

54 165 0.01 7 43 5.1 1 *

12 42 0.22 166 5 62 1.88

* Significant at the 5% level.

BROWDER (1968) reported a significant excess of Burnsi progeny from a cross between an extreme speckle, burnsi male and a normal R. pipiens female. The most striking thing about this set of data (34 ES; 25 normal; 54 Burnsi; 45 ES, Burnsi) is the deficiency of the wild type. The Burnsi: non-Burnsi ratio is significantly different from 1 : l ( x 2 = 10.17; P < 0.01). More recently, BROWDER (in Zitt.) has obtained a significant excess (P < 0.01) of non-Burnsi progeny from descendants of this cross. One possible explanation for these seemingly anomalous results is that a deleterious gene linked to the normal allele of B became associated with the B gene by crossing over, thus accounting for the excess of Burnsi animals in the early cross and the excess of non-Burnsi in the more recent cross.

Tables 3 and 4 give the results of crosses involving Kandiyohi: Table 3 (K/+) x (+/+) and Table 4, ( K / f ) X (K/+) . Again the results do not differ significantly from the expected 1 : 1 and 3: 1 ratios.

Table 5 gives the results of crossing heterogygous Burnsi (B+/; +/+) with heterozygous Kandiyohi frogs (-I-/+; K / f ) and also the double heterozygotes ( B / + ; K / f ) with the wild type (+/+; +/+). VOLPE (1960) first showed that equal numbers of +, Kandiyohi, Burnsi, and Burnsi-Kandiyohi frogs were ob-

TABLE 3

Crosses between Kandiyohi (K/f ) and wild type (+/+)

Investigator

MERRELL RODELL* VOLPE (1955) K x f

+ X K VOLPE (1970) ANDERSON and VOLPE (1958) K x f NACE et al. (1970) Total

~ ~ ~ ~ _ _ _ _ _ _ _ ~

Progeny (N) +/+ W+ X=

131 133 0.02 94 117 2.52

12.3 117 0.15 100 118 1.48 52 46 0.36

111 91 1.98 94 88 0.20

705 710 0.02

* Data kindly furnished by CHARLES F. RonmL, Dept. Zoology, Univ. Minnesota.

P I G M E N T P A T T E R N S IN FROGS

TABLE 4

Crosses between Kandiyohi heterozygotes (K/+ x K/+)

145

Progeny (N) Investigator +/+ K/- X=

MERRELL 4 6 . . . VOLPE (1955)

(1956) NACE et al. (1970) Total

57 197 0.89 49 171 0.87 21 59 0.05

131 433 0.95

tained from the Burnsi-Kandiyohi x wild type cross, demonstrating that the B and K genes are not linked. Therefore, the expected ratios are the same for both types of cross, and the data can be grouped.

For the grouped data from I and I1 of Table 5, x p is significant at the 5% level when the 1 : 1 : 1 : 1 ratio is tested. If all four types had equal viability, the probability that any one type would be the least frequent in any given experiment is one in four. The probability that the same type would be the least frequent in five separate experiments is ( x ) or only 1 in 1024. Since the Burnsi-Kandiyohi type was least frequent in each of these experiments, the explanation for the deviation from the 1 : 1 : 1 : 1 : ratio in these experiments appears to be a slight but significant reduction in viability of the double dominant heterozygote. The ex- pectation for this type of frog, because of the low frequency of both the B and the K genes (MERRELL 1965), is very low in natural populations in the area of over- lap (of the order of 1 in 10,000). Hence, unlike wild type, Burnsi, and Kandiyohi, the Burnsi-Kandiyohi phenotype is rarely exposed to natural selection.

The recent data of NACE, RICHARDS and ASHER (1970) and the data of VOLPE

TABLE 5

Crosses involving Burnsi and Kandiyohi

Progeny (N) Investigator + K B BK

I B X K MERRELL 140 115 113 108 5.15 A N D E R S O N ~ ~ ~ V O L P E (1958) 57 65 54 51 1.91

VOLPE (1960) 45 37 46 30 4.28 VOLPE ( 1 970) 35 30 33 27 1.18 Total 281 253 251 217 8.39+ NACE et al. (1 970)

I1 BK X + MERRELL 4 6 5 1 , . .

111 9 K X $ B 48 49 42 47 0.60 9 B X $ K 61 35 65 42 12.461

9 K X $ B 67 64 117 . . 0.86 9 B X $ K 57 69 128 . . 1.16

+ B K + BK$ x2?df IV VOLPE (1956)

* Significant at the 5% level. t Significant at the 1 % level. $ K and BK were not distinguished.


(1956) are listed separately because of the anomalous results of NACE et aZ. with the Burnsi 0 x Kandiyohi 8 crosses and because VOLFE did not distinguish between Kandiyohi and Kandiyohi-Burnsi progeny. The deficiency of Kandiyohi progeny in the Burnsi 0 x Kandiyohi 8 crosses of NACE et al. are suggestive of BROWDER’S results with Burnsi mentioned earlier and may have a similar explanation. None of the other crosses involving Kandiyohi shown in Tables 3,4, and 5 show a deficiency of Kandiyohi.

DISCUSSION

BROWDER’S results (1967) rearing homozygous recessive albino frogs stand in contrast to the results obtained with the Burnsi frogs. His frogs were reared in this laboratory under conditions comparable to those used with Burnsi. Among the young F, tadpoles the ratio of wild type to albino progeny was not significantly different from 3: 1. However, in the tadpoles selected for rearing, a highly significant difference between albinos and wild type was found in the incidence of scoliosis. Only 6 of 43 wild-type tadpoles scored showed the characteristic bend of the body at the ba:e of the tail, but 35 of 41 albino tadpoles ccored were scoliotic. Of the 50 wild-type (A/--) tadpoles selected for rearing, 43 survived to forelimb emergence; of 41 albinos ( a l a ) , 28 survived to forelimb emergence, a difference that is not statistically significant. However, among the frogs selected for rearing following metamorphosis, only 4 of 19 albinos survived past the 21st week after metamorphosis while all seven wild-type frogs survived, a highly significant difference in viability (see also UNDERHILL 1966). Therefore, the evidence on scoliosis in tadpoles and viability in young frogs indicates that the albino gene, when homozygous, has a detectable deleterious effect under rearing conditions comparable to those used in the experiments reported here. BROWDER’S results suggest that if the Burnsi or Kandiyohi genes had harmful effects, these effects would have been detected. GILL (1970) has recently reported that embryos derived from K/+ mothers had significantly higher survival during embryogenesis than those from +/+ or B / + females, regardless of paternal qenotype, but that significantly more K/+ offspring from K/+ X +/+ reciprocal crosses died during metamorphic climax than their +/+ sibs, yielding a final ratio of K/+ to +/+ juveniles of 1: 1. The balance between differential viability of embryos from K/+ females and differential mortality of K/+ heterozygotes during late metamorphosis is postulated as contributing to the maintenance of the Kandiyohi polymorphism. Furthermore, she reported that reciprocal crosses of B/+ x +/+ yielded a significant excess of B / + progeny at all late metamorphic stages, and that the ratios of Burnsi to wild type were not significantly different from those in B/+ x B / + crosses. Her results with Burnsi are certainly at odds with the data shown in Tables 1 and 2. Since no data are given in the abstract, it is difficult to evaluate her Kandiyohi hypothesis.

B. DEVELOPMENTAL RATE O F MUTANT AND WILD PHENOTYPES

Although the data showed no evidence that the mutant frogs were less viable than the wild type, except for the slight reduction in viability of the double Burnsi-Kandiyohi heterozygote,

PIGMENT PATTERNS I N FROGS

TABLE 6

147

Comparison of mean snout-vent lengths (mm) of newly metamorphosed frogs, progeny of the same cross reared together

Cross Phenotype

B x + + B

K X f + K

N Z - C S ~ N Z + S ~ 264 26.6 f 0.2 229 26.9 2 0.2

66.13 96 25.0 -C 0.3 108 24.7 k 0.3 RODELL 94 26.2 & 0.3 117 26.6 2 0.2

B X K + B K BK 86 25.0 -t 0.4 75 24.6 + 0.3 70' 23.8 2 0.4 71* 24.5 -C 0.4

* 1 BK and 2 K frogs not included due to death prior to complete metamorphosis.

the possibility remained that the genes might have other effects. Records were kept of the date metamorphosis was completed, specifically the time at which the tail was completely resorbed, and of the snout-vefit length at that time.

RESULTS

Table 6 contains the data on snout-vent length of newly metamorphosed frogs from the same cross reared together. Only one significant difference appeared, that between wild-type and Kandiyohi progeny from the Burnsi x Kandiyohi cross where a t test gave 0.02 > P > 0.01. However, no such difference was observed in the two crosses of Kandiyohi and wild type so that it seems safe to con- clude that genotype had no significant effect on snout-vent length in newly metamorphosed frogs in these experiments.

However, the data shown in Table 7 and Figures 1-3 show that in three separate experiments in which large numbers of progeny were available, Kandiyohi tadpoles completed metamorphosis significantly earlier, on the average, than the other genotypes being reared in the same tanks.

The means and standard errors of age at metamorphosis were obtained by the SPEARM.4N-KiRBER method (FINNEY 1964). As can be seen in the figures, the

TABLE 7

Mean age at metamorphosis in days ( SPMRMAN-KARBER)

Cross Wild type Kandiyohi

N X - C S ; N Z + S ; 66-13 96 133.9 2 1.5 109 122.4 k 1.3* 67 RODELL 94 145.1 -C 1.6 117 135.5 t 1.3*

Wild type Kandiyohi Burnfi Burnsi-Kandiyohi N Z t s ; N x f s ; N x f s ; N

66.14 86 101.3 k 1.2 72 94.0 k 1.2* 75 101.5 k 1.2 72 103.5 + 1.3

* P < 0.01.

148

100-

90

80

70

60- 0 2 50- cc

40- U b 30- 2 ap 20-

IO-

DAVID J. MERRELL

- P vs. K -

-

-0 + o s *& S a , ,

I I I I I O,C'*'iY I I I I I I I I

relation between age and percent metamorphosed is not strictly linear, but trans- formation to log days did not appreciably improve the situation. Therefore, the estimates were made using days rather than log days, and must be regarded as rough approximations. Nevertheless, the difference between Kandiyohi and the

lOOr

90 -

80

70

P vs. K - -

8 40-

* N O

0 IO 20 30 40 50 60 70 80 90 100 110 120 130 1409 $ DAYS

FIGURE 2.-Time of metamorphosis of progeny from cross of 0 wild type (f/+) X 8 Kandiyohi ( K / + ) made on 4-14-67 by C. F. RODELL. Day 0 in Figure is 7-8-67. For wild type progeny, n=94; for Kandiyohi progeny, n=ll7. = wild type; 0 = Kandiyohi.

PIGMENT PATTERNS IN FROGS 149

0 IO 20 30 40 50 60 70 80 90 100 110 120 130 140 DAYS

FIGURE 3.-Time of metamorphosis of progeny from cross 9 Burnsi ( E / + ; +/+) X 8’ Kandiyohi (+/+; K / + ) made on 4-14-66. Day 0 on Figure is C20-66. For wild-type progeny, n d 6 ; for Kandiyohi, n=72; for Burnsi, n=75; for Burnsi-Kandiyohi progeny, n=72. = wild type; 0 = Kandiyohi; A = Burnsi; A = Burnsi-Kandiyohi.

other genotypes in rate of development appears to be real. VOLPE (1955), however, measuring the rate of development in embryos, reported no difference between Kandiyohi and wild-type embryos.

N:, marked difference in developmental rates between Burnsi and wild type is revealed by the data in Figure 3 olr by other data invoslving only B / + (N = 229) and +/+ ( N = 264) progeny.

DISCUSSION

The above data on Kandiyohi appear to suggest that the K/+ heterozygotes have a faster rate of development than the other genotypes tested. It was reported earlier (MERRELL 1965) that the Kandiyohi gene is associated with the prairie regions of western hlinnesota and the eastern Dakotas. ZWEIFEL (1968), in a study of nine species of anuran amphibians in the arid Southwest of the United States, found “a clear correlation between the rate of development and the length of time the breeding sites may be expected to retain water.” Because of the spo- radic pattern of rainfall on the prairies. a rapid rate of development would confer an adaptive advantage there as well. Moreover, since temperature tolerance broadens as embryonic development proceeds, a more rapid rate of development may also help to protect the developing frog from the extremes of temperature to be found in prairie breeding ponds. Therefore, it may be postulated that one adaptive advantage of the Kandiyohi gene in the prairie habitat lies in the more rapid rate of development of frogs carrying this gene, which, in times of drought,


may enable them to complete metamorphosis before the breeding ponds dry up. This interpretation is supported by field observations. Even in eastern Minne-

sota, where rainfall is normally greater than further west on the prairies of western Minnesota and the Dakotas, breeding ponds have been observed to dry up completely in a dry spring before the Rana pipiens tadpoles in these ponds could complete metamorphosis. Furthermore, the large annual fluctuations in numbers of frogs sometimes observed can be related to the availability and per- sistence of breeding ponds, which vary from year to year, sometimes quite markedly (MERRELL 1965).

11. CROSSES IKVOLVING SPOT NUMBER

Analysis of the data from frogs collected in Minnesota and adjacent areas indicated that the wild-type R. pipiens frogs found within the range of Burnsi had fewer spots between the dorsolateral lines than the wild-type frogs living both north and south of the range of Burnsi (MERRELL 1965). This finding raised questions about the nature of the influences on the spotting pattern in the usual wild-type phenotype. Unlike the relatively simple genetics of the Burnsi and Kandiyohi genes, the situation in this case is more complex. The nature of the trait, dorsal spot number, merits some attention. It is a meristic character, and can be treated, therefore, as a continuously varying quantitative trait. Strictly speaking, of course, it is not, because the spots are measured in whole numbers. However, the potential for spot production can be regarded as a continuously varying character that can only be expressed in whole numbers. As with other quantitative traits, it seems safe to assume that a number of different segregating gene loci affect the character so that the effects of the individual genes are difficult to identify. Furthermore, the trait might be expected to vary because of non-genetic effects, which ordinarily vary continuously and hence tend to obliterate any genetic discontinuities that may arise due to the segregating genes. Therefore, crosses were made to investigate possible genetic and environmental influences on spot number. (VOLPE and DASCUPTA 1962; DAVISON 1964; MERRELL 1965).

In order to count the spots between the dorsolateral lines, certain conventions had to be adopted (MERRELL 1965). All visible spots were counted. If only part of a spot lay within the area bounded by the dorsolateral lines, more than half a spot was counted as one spot, but anything less was not counted. Sometimes two spots appeared fused. If there were detectable indentations on both sides, it was counted as two (or in some cases more) spots. If indentations were not present or were found only on one side, it was counted as just one spot. This method gave consistent results with different observers. Spot number rather than spotting pattern was studied because it is more readily scored and treated statistically. Biologically, each spot seems to represent a focus for the concentration cf pigment cells.

A. CONSTANCY OF SPOT NUMBER

It is axiomatic that a leopard cannot change its spots and BRFSLFX (1963), DAVISON (1964), and UNDERHILL (1968) are agreed that this is also true of the leopard frog, once metamorphosis is completed. However, DAVISON and UNDERHILL did not present data, and BRESLER’S conclusion was based on inference. BRESLER defined a spot as “black pigmentation at least one millimeter in length or width.” After grouping frogs into three size classes, he found no significant differences in spot number between small, medium and large frogs. However, he did find that the percentage of the total area covered by spots actually increased somewhat with increasing body length.

RESULTS

As a check on the constancy of the spot number and on the reliability of the method used in counting spots in these studies, fourteen newly metamorphosed frogs were individually marked by toe clipping and their spots were counted at


TABLE 8

Dorsal spot counts of individually marked frogs during growth (Date of cross-4/12/65)

Date Frog No. 8/16/65 12/30/65 4/6/66 6/1/66 6/29/66 10/6/66

1 L 31 53 63 63 67 74 N 11 12 12 12 12 12

2 L 32 40 45 56 59 60 N 12 11 11 11 12 12

3 L 34 48 59 75 80 82 N 13 13 13 13 12 12

4 L 34 48 52 66 69 t N 12 12 12 12 12 . .

6 L 38 47 58 65 65 69 N 17 17 17 19 19 19

7 L 40 52 56 71 78 t N 15 15 15 16 16 . .

8 L 40 45 48 62 65 t N 13 13 13 13 13 . .

9 L 39 47 51 65 t . . N 12 12 12 12 . . . .

11 L 33 4 4 50 54 57 59 N 13 13 13 13 13 13

12 L 35 4 6 52 63 66 71 N 13 13 13 15 15 15

N 9 9 9 . . . . . . 14 L 37 46 54 62 63 64

N 12 12 12 12 12 12

. . . . 13 L 32 39 38 t

L=Snout-vent length in mm. N=Number of spots between the dorsolateral lines.

intervals during growth. These frogs were F, progeny from a cross between a male pseudokandiyohi (see MERRELL 1965 ) and a wild-type female, being reared to sexual maturity for use in F, crosses. The data on 12 of these frogs are set forth in Table 8. The seven frogs that survived the entire period doubled in length, but average spot number appeared to increase only about half a spot. Some frogs had an unchanged spot count throughout the experiment; in others, fluctuations can be seen in the counts, decreases as well as increases. These fluctuations are due not to changes in the number of spots on the frogs but to difficulties in interpretation of the patterns. In some frogs complex spots or small spots or both made conversion of the spotting pattern into a dorsal spot count more subjective than usual. If anything, these difficult patterns seemed to be more common among this group of lab-reared irogs than among frogs in natural populations and may be related to the fact that the male parent was classed as a pseudokandiyohi. Never- theless, the margin of error stemming from difficulties in interpreting the pattern was not particularly great, and it seems clear that the leopard frog is indeed un- able to change its spots.


B. Crosses involving spot number.

The results from crosses involving spot number are shown in Tables 9 and 10. In Table 9, it can be seen that the newly metamorphosed progeny from crosses between parents with low spot numbers do not differ significantly from their parents in average spot number. The parental mean given is an unweighted mean for which a standard error was easily calculated. Since the table shows that some parents contributed appreciably more progeny than others, a weighted mean was also calculated. This value is 10.2, not greatly different from the unweighted valur? of 9.9. For the parcnts in Table 10, the weighted mean was 20.2, as com-

TABLE 9

Crosses between parents with low numbers of dorsal spots

hIean spot nuniber Parental Mean parental Progeny of progeny

spot number spot Ilunlber N s * s; Range

lox 8 9.0 6 13.5 zk 1.0 10-16 11 x 11 11.0 77 11.0 i 0.2 7-16

1 1 x 8 *

l l x 8 * 11 x 11 lox 11 7x 11

Parents N=18 9.9 f 0.4s 265 10.6 f 0.1

1 1 x 7 9.0 29 9.7 f 0.3 7-12

10.2 1141 10.4 i 0.2 6 1 6 11 x 111 } 10.0 3% 10.5 f 0.4 6-16

* From MERRELL (1965). t Tadpoles reared at approximately 25 f 1°C. $ Tadpoles reared at approximately 20 t 1°C. $ Unweighted mean. See text.

TABLE 10

Crosses between parents with high numbers of dorsal spots

Mean spot number Parental Mean parental Progeny of progeny

spot number spot nuniber l< s i s; Range

20 l7 x 211 17 18.8 38 11.6 f 0.3 9-18

22 x 40’ 31.0 65 12.8 zk 0.2 9-17 16 x 20 18.0 110 13.6 zk 0.2 1 9 x 26 22.5 150 13.7 i 0.1 11-18

1 O s t 12.8 f 0.2 8-20 69$ 14.2 k 0.3 7-21

541 13.3 i 0.1 18 x 14* {;E Parents N=12 20.8 i 2.0s

* From MERRELL (1965). f Tadpoles reared at approximately 25 f 1°C. $ Tadpoles reared at approximately 20 i- 1°C.

Unweighted mean. See text.


pared to the unweighted 20.8. Hence, use of the unweighted means and their standard errors would seem unlikely to bias the results to any significant extent. In addition, the frogs reared at 20°C (Table 9) do not differ significantly in spot number from those reared at 25°C.

Table 10, however, shows the progeny of parents with high spot numbers to have significantly fewer spots than their parents. Furthermore, the progeny reared at 25°C had significantly fewer spots than those reared at 20"C, in contrast to the results in Table 9.

Nevertheless, a comparison of the progeny of parents with low spot numbers with the progeny of parents with high spot numbers reveals that these two groups show a highly significant difference in average spot number (10.6 -+: 0.1 us. 13.3 0.1; P < 0.001). Therefore, these results indicate an hereditary influence on spot number (see also CASLER, 1967).

DISCUSSION

Heritability: The reason for the difference in the amount of regression toward the mean in the high and low crosses is not readily apparent. One possible factor may be that the environmental rearing conditions tended to favor the production of frogs with relatively low spot number, whatever their genotype. Another possible explanation is that the lower limit for spot number for wild-type R. pipiens in natural populations in the Minnesota area is about 7, and the mean for the populations in East Central Minnesota is about 13 to 14 spots. However, there appears to be no well-defined upper limit for spot number comparable to the lower limit. Therefore, the possible range of variation for frogs on the low side of the mean is considerably less than for those on the high side.

UNDERHILL (1968) estimated the heritability of dorsal spot number in Rana pipiens to be 0.14 so that the nature of the results in Tables 9 and 10 is perhaps not surprising. Heritability is defined as the ratio of the additive genetic variance to the phenotypic variance. However, the heritability can also be estimated as the ratio of response to selection over the selection differential. In this case the selection differential may be taken as the difference between parental means (20.8 - 9.9 = 10.9) and the response as the difference between the means of the progeny (13.3 - 10.6 = 2.7). The heritability estimate, then, is

n v - L.1 10.9

h - 0.25.

which is in fairly good agreement with UNDERHILL'S estimate of 0.14. This somewhat unorthodox method of estimating heritability is used because no meaningful estimate of a population mean could be made. Different frog populations have been shown to have widely different average numbers of spots (MERRELL 1965), and the average spot numbers of the populations from which the selected frogs came are unknown.

These relatively low heritability estimates are indicative of little additive genetic variance. In contrast to the results in MERRELL (Table 3, 1965), which indicate that the number of dorsal spots is independent of body size at metamorpho-


sis, UNDERHILL’S data suggested that the larger the offspring at metamorphosis, the greater the number of dorsal spots. In addition, BROWDER has reported (in litt.) that “when animals are reared under extreme conditions of crowding during early larval life followed by a release from the crowded conditions to optimal conditions, both size and spot number are reduced. It is a safe generalization (h my laboratory) that the first animals to metamorphose in nearly all crosses (analogous to crowding) are smaller than the last ones. Thus, spot number would be expected to increase as number of metamorphosed animals increases.” UNDER- HILL’S data resembled those in Table 10 in that the mean spot number in the progeny was significantly lower than that of the parents. All of these results agree in suggesting that heredity plays a role in the determination of dorsal spot number but that non-genetic influences are also of considerable importance.

Nuclear transplantation: Another type of experiment bearing on the nature of the control of the pigment pattern in Rana pipiens involves nuclear transplantation and the formation of clones of genetically identical frogs (MCKINNELL 1962; CASLER 1967). Fertilized eggs were allowed to develop to the blastula stage and then the blastula nuclei were transferred to enucleated eggs, which are then per- mitted to develop through metamorphosis. In some cases, in order to obtain larger groups within a clone, serial nuclear transfers were carried out, that is, a blastula that developed from a nuclear transfer served as the nuclear donor for still another group of enucleated eggs.

CASLER (1967) compared the pigment patterns of clones of individuals with the patterns of individuals arising from normally fertilized eggs, and found no significant difference in spot number, average spot size, or spot location and shape. However, individuals within clones were more alike in the percentage area covered by spots than the controls. As in the experiments reported above in Tables 9 and 10, she found that progeny of parents with high spot number had significantly more spots lhan the progeny of parents with lower spot numbers.

In contrast to CASLER’S results, MCKINNELL (1962) found through an analysis of variance that the differences in spot number among clones were greater than the average differences among individuals within a clone. In other words, the individuals within a clone were more uniform in spot number than might be expected among individuals selected at random from the population. Nevertheless, within a given clone, all with identical genotypes, spot numbers were not all identical. No obvious explanation for the difference in the outcome of the transplantation experiments of CASLER and NICKINNELL is apparent from the data presented. One possibility may be that CASLER’S controls were exceptionally uniform. However, these results do support the idea that both hereditary and environmental effects are responsible for the pigment pattern.

Genetic and environmental influences on the pigment pattern: The results of DAVISON and of VOLPE also support this concept although their interpretations are rather different. VOI.PE (1961) attributed variations in the amount of spotting in Burnsi heterozygotes to the action of modifying genes at other loci. BROWDER and DAVISON (1964), on the other hand, felt that with one exception, the variations they observed were due to environmental effects. The exception they re-


garded as due to “a different locus, allele or pseudoallele” rather than the effects of modifiers.

At this point it should be noted that the Burnsi and Kandiyohi genes are es- sentially pattern mutations whose primary effect seems to be on pigment cell d;stribution rather than on the presence or absence of any of the three types of pigment cells (see also GILL and NACE 1969). All three types of pigment cells (melanophores, iridophores, and xanthophores) are present in Burnsi and Kandi- yohi frogs. In essence, dorsal Burnsi skin resembles the dorsal skin from the background region between the spots in the wild type (BAKER 1951). The spots in wild-type R. pipiens, however, have a greater total amount of melanin, more melanin per melanophore, more intracutaneous melanophores, but fewer epi- dermal melanophores than the wild-type background skin or dorsal Burnsi skin. BAKER found, in addition, that wild-type skin in both the spotted and background regions had a higher tyrosinase activity than Burnsi skin. However, it should be remembered that Burnsi frogs normally have spotted legs and occasionally dorsal back spots as well, and hence are capable of spot formation. The relationship, if any, between tyrosinase activity and spot formation has never been established. Studies comparable to BAKER’S with Burnsi have never been made with Kandi- yohi.

The Burnsi and Kandiyohi phenotypes should be compared with the albino, blue, speckle, and extreme speckle types studied by BROWDER (1967,1968). The albino frogs lack pigmented melanophores. Nevertheless, pigmented iridophores and xanthophores are both present and the normal spotting pattern appears as orange spots on an ivory background. Thus, functional melanophores are not necessary for the production of the wild-type spotting pattern. Blue frogs have iridophores and melanophores but lack pigmented xanthophores. They too show the wild-type spotting pattern. Albinism is due to a homozygous recessive gene, and blueness is probably the result of a different recessive. The blue frog referred to here is completely blue and differs from the blue and green non-hereditary type reported on by BERNS and NARAYAN (1970). The speckle and extreme speckle variants are produced by two dominant genes of variable expression that may or may not be alleles. The speckle homozygotes also show the wild-type spotting pattern. Electron microscope studies (BROWDER and WISE 1970; BROWDER, in litt.), have shown that iridophores are absent. Therefore, since the spotting pattern appears in the absence of functional cells of each of the three types of pigment cells (Table 1 1) , the pattern is evidently not dependent on the presence of functional representatives of any one type of chromatophore. This point is worth making because the assumption has been explicitly made that the pigment pattern is dependent on “the potentialities of melanoblasts” (VOLPE 1964) or on “spot-forming melanophores” (VOLPE 1963, see also VOLPE and DASGUPTA 1962). This assumption is understandable, for the spots normally result from a concentration of melanophores and melanin in a given area. However, the albino frogs have the normal spotting pattern despite the absence of melanin so that functional melanophores are not essential for spot formation.

However, transplantation experiments with Burnsi or Kandiyohi or double


TABLE 11

Pigment cell types in albino, blue and speckled Rana pipiens. (After Browder)

Pigmented Unpigmented Cell types Phenotype cells present cells present absent

Albino Iridophores Melanophores Xanthophores

Blue Iridophores Xanthophores Melanophores

Speckle Melanophores Iridophores Xanthophores

mutant frogs show the donor pattern when the neural crests are exchanged (DAVISON 1963). DAWSON interprets these results to mean that the Burnsi and Kandiyohi genes act “through modifications in the pigment-forming elements themselves, rather than in the skin substrate through which these cells aggregate and differentiate to determine the adult phenotype.” Since the normal pigment pattern appears despite the absence of functional melanophores, or iridophores, or xanthophores, the Burnsi and Kandiyohi genes must have an influence on all of the chromatophores or neural crest derivatives in order to produce the pattern, if DAVISON’S interpretation is correct.

One assumption implicit in the discussion of the Burnsi and Kandiyohi pattern genes should be made explicit. It seems a reasonable assumption that the genetic or environmental factors affecting the spotting pattern in the mutant frogs will also influence the spotting pattern in the wild-type frogs.

In support of their position on the importance of environmental effects, DAVI- SON (1961 ) and BROWDER and DAVISON (1 964) have reported that Burnsi heterozygotes reared at high temperature (30°C) and reduced oxygen tension were unspotted and that the amount of spotting increased with decreased temperature so that at 18”C, frogs with this genotype had spotted legs plus a few back spots. DAVISON also reported (1964) that he obtained phenocopies of the Burnsi type in genetically wild-type animals reared at high temperatures. Moreover, he showed (1964) that members of each of seven pairs of Burnsi heterozygotes reared in parabiosis had patterns very similar to one another. Finally, heterozygous sibs undergoing metamorphosis at the same time were more alike than those metamorphosing at different times. All of these results are in accord with the idea that the pigment pattern can be markedly influenced by environmental conditions.

DAVISON (1961) also studied the effect of gene dosage on the pigment pattern in diploid and triploid frogs. He reported that increasing dosage of the Burnsi gene decreased the amount of spotting. Although he determined diploidy and triploidy by cytological examination, he apparently determined the dosage of the Burnsi gene by inference from the frogs’ phenotypes. In view of his own results demonstrating the sensitivity of the pigment pattern to environmental influences, this procedure seems open to question, especially since the effect of gene dosage was his primary concern in these experiments. Even though his inferences may

PIGMENT PATTERNS I N FROGS 157

havz been correct, the most reliable method to determine the genotypes of the progeny would have been to rear them to sexual maturity and make appropriate test crosses, difficult though this would have been. That his inferences may have been incorrect is suggested by the fact that the animals scored as being in the crossover classes far exceeded expectations.

MOORE (1942) and DAVISON (1961 ) reported that they could distinguish between B / + and B / B progeny from the cross B / + x B / + . Supposedly, B / + has some leg spots, which are virtually lacking in BIB. For this reason, MOORE suggested that the Burnsi gene is not completely dominant. Since VOLPE and DAS- GUPTA (1962) in B / + x B / + crosses between heavily spotted heterozygotes got only rather well spotted frogs, it seems preferable to suggest that Burnsi shows a variable degree of dominance. Since one third of the Burnsi frogs in this cross must have been homozygous B/B, these results seem to show that B / B as well as B / f frogs can produce spots. Furthermore, spotless frogs have also been shown to be heterozygous B / + in genotype. Hence, at present it seems advisable to re- serve judgment as to whether under similar environmental conditions B / B frogs will consistently have fewer spots than B / + frogs.

In general, BROWDER and DAVISON (1964) found that spot number in the progeny was unrelated to spotting in the parents in Burnsi ( B / + ) x wild type (-I-/+) crosses, but in one case a spotless Burnsi male produced only spotless or lightly spotted progeny. The result of this experiment resembles VOLPE and DASGUPTA’S results (1962), which showed that unspotted Burnsi parents tended to have lightly spotted progeny while heavily spotted Burnsi parents tended to have heavily spotted Burnsi progeny. For their exceptional case, BROWDER and DAVISON postulated either a different allele of B, a pseudoallele, or a gene at an entirely different locus. VOLPE and DASGUPTA attributed their results to the effects of plus and minus modifying genes on the expression of the Burnsi gene.

The ad hoc postulation of a different B allele is probably the simplest possible explanation for BROWDER and DAVISON’S atypical result. However, the known sensitivity of Burnsi expression to environmental influences leaves open the possibility that this result might have been due to the rearing conditions. Further- more, the limited distribution and brief history of the Burnsi gene (MERRELL 1965, 1970) suggest that Burnsi alleles from the Minnesota area are identical by descent and are not an assortment of isoalleles.

VOLPE’S theory of modifying genes requires comment also, for he postulates that the modifying factors are of “negligible” effect (VOLPE and DASGUPTA 1962) and “virtually undetectable” (VOLPE 1961) in the wild type (+/+). The fact is, of course, that since the Burnsi gene is rather rare in natural populations, the modifying genes, if they exist, will have been selected in relation to the wild-type phenotype rather than Burnsi (contrary to VOLPE 1961). While they may affect the expression of the Burnsi gene, this effect is incidental to their role in en- hancing or decreasing the amount of spotting in the wild phenotype. This interpretation is supported by UNDERHILL’S study (1968) on the heritability of spot number, by CASLER’S data (1967), and by the results from wild type X wild type crosses in Table 9 and 10 showing progeny from parents with high spot numbers


to have significantly more spots than progeny of parents with low spot numbers, and by the finding (MERRELL 1965) that wild-type frogs within the range of Burnsi have fewer spots than the wild type outside the Burnsi range. This result need not be attributed to a “Burnsi influence” (BRESLER 1963), which suggests an effect of Burnsi itself on the wild-type spotting pattern. Instead, it suggests that natural selection, within the range of Burnsi, is acting to reduce the amount of spotting in the frogs by two different genetic mechanisms. In one case, the reduction is mediated by the dominant Burnsi gene; in the other, selection appears to have favored modifying factors that reduce the amount of spotting in the wild phenotype.

Because VOLPE considered the effect of the modifying factors undetectable in the wild-type frogs. he unfortunately did not present information on the spot numbers of the wild-type parents and progeny in his crosses. Since the wild-type parent must contribute modifiers equally with the Burnsi parent, it is essential to have some knowledge of its phenotype. Similarly, information about the wild- type progeny should be as instructive as information about the Burnsi progeny with respect to any theory about modifying genes.

The able assistance of CHARLES F. RODELL is gratefully acknowledged. Drs. LEON W. BROWDER and ROBERT G. MCKINNELL very kindly read the manuscript and made helpful comments. This work has been supported in part by the National Science Foundation (GB 3663) and the Graduate School of the University of Minnesota.

LITERATURE CITED

ANDERSON, S. C. and E. P. VOLPE, 1958 Burnsi and Kandiyohi genes in the leopard frog, Rana pip iens . Science 127: 1048-1050.

BAKER, A. S., 1951 A study of the expression of the Burnsi gene in adult Rana pip iens . J. Exptl. Zool. 116: 191-229.

BERNS, M. W. and K. S. NARAYAN, 1970 An histochemical and ultrastructural analysis of the dermal chromatophores of the variant ranid blue frog. J. Morphol. 132: 169-180.

BRESLER, J. B., 1963 Pigmentation characteristics of R a m pip iens: Dorsal region. Am. Mid . Nat. 70: 197-207.

BROWDER, L. W., 1967 Pigmentation in Rana pip iens: A study in developmental genetics. Ph.D. Thesis, University of Minnesota. --, 1968 Pigmentation in Ram pip iens: I. Inheri- tance of the speckle mutation. J. Heredity 59: 162-166.

BROWDER, L. W. and J. DAVISON, 19W Spotting variations in the leopard frog. A test for the genetic basis in the R a m pip iens ‘‘burmi” variant. J. Heredity 55: 234-241.

BROWDER, L. W. and G. E. WISE, 1970 The ultrastructure and biochemical composition of the skin of the speckle mutant of the leopard frog, R a m pip iens . (Abstr.) Can. J. Genet. Cytol. 12: 375.

CASLER, E., 1967

CLAUSEN, J. and W. M. HIESEY, 1958

DAVISON, J., 1961

Pattern variation in isogenic frogs. J. Exptl. Zool. 166: 121-136.

Experimental studies on the nature of species. IV. Genetic structure of ecological races. Carnegie Inst. Wash. Publ. 615: 1-312.

A study of spatting patterns in the leopard frog. I. Effect of gene dosage. J. Heredity 52: 301-304.. -- , 1963 Gene action mechanisms in the determination of color and pattern in the frog ( R a m p i p i e n s ) . Science 141 : 648-64.9. 1964. A study of spotting patterns in the leopard frog. 111. Environmental control of genic expression. J. Heredity 55: 47-56.

-,


DOBZHANSKY, TH., 1970 Genetics of the evolutionary process. Columbia, New York. FISHER, R. A., 1939 Selective forces in wild populations of Paratettix texanus. Ann. Eugenics

9: 109-122. FORCE, E., 1933 The age of attainment of sexual maturity of the leopard frog Rana pipiens

(Schreber) in northern Michigan. Copeia 1933: 128-131.

FORD, E. B., 1964 Ecological genetics. Wiley, New York. GILL, S. J., 1970 Differential viability associated wit hthe Burnsi and Kandiyohi heterozygotes

in the leopard frog, R a m pipiens. (Abstr.) Genetics 64 (Suppl.) : 23-24. GILL, S. J. and G. W. NACE, 1969 Pigment distributions in pattern mutants of Rana pipiens.

(Astr.) Am. Zool. 9: 1125. KETTLEWELL, H. B. D., 1961 The phenomenon of industrial melanism in the Lepidoptera. Ann.

Rev. Ent. 6: 245-262. Intraspecific nuclear transplantation in frogs. J. Heredity 51 : 199-207.

Rearing tadpoles of the leopard frog, R a m pipiens. Turtox News 41: 263-265. --, 1965 The distribution of the dominant Burnsi gene in the leopard frog, R a m pipiens. Evolution 19: 69-95. - , 1968 A comparison of the estimated size and the “effective size” of breeding populations of the leopard frog, Rana pipiens. Evolution 22: 274-283. -, 1969a Limits on heterozygous advantage as an explanation of polymorphism. J. Heredity 60: 180-182. --, 19691, Natural selection in a leopard frog population. J. Minn. Acad. Sci. 35: 86-89. - , 1970 Migration and gene dispersal in Rana pipiens. Am. Zoologist 10: 47-52. -, in press Studies in ecological genetics with Rana pipiens.

MERRELL, D. J. and C. F. RODELL, 1968 Seasonal selection in the leopard frog, R a m pipiens. Evolution 22 : 284-288.

MOORE, J. A., 1942 An embryological and genetical study of Rana burnsi Weed. Genetics 27: 408-416. -, 1943 Corresponding genes in spotted frogs of the genus Rana. J. Heredity 34: 3-7.

MCKINNELL, R. G., 1962

MERRELL, D. J., 1963

NABOURS, R. K., 1929

NACE, G. W., C. M. RICHARDS and J. H. ASHER, 1970 Parthenogenesis and genetic variability. I. Linkage and inbreeding estimates in the frog, R a m pipiens. Genetics 66: 3W-368.

PASTERNACK, B. S. and R. R. GIANUTSOS, 1969 Application of the exponential and power func- tions to the study of allometric growth, with particular reference to doryline ants. Am. Naturalist 103: 225-234.

RICHARDS, C. M., 1958 The inhibition of growth in crowded R a m pipiens tadpoles. Physiol. Zool. 31: 138-151.

ROSE, S. M., 1960 A feedback mechanism of growth control in tadpoles. Ecology 41: 188-199.

RYAN, R. A., 1953 Growth rates of some ranids under natural conditions. Copeia 1953: 73-80.

SHUMWAY, W., 1940 Stages in the normal development of R a m pipiens. I. External form.

UNDERHILL, D. K., 1966 An incidence of spontaneous caudal scoliosus in tadpoles of Rana pipiens Schreber. Copeia 3: 582-583. - , 1968 Heritability of dorsal spot number and snout-vent length in R a m pipiens. J. Heredity 59 : 235-240.

A taxo-genetic analysis of the status of R a m kandiyohi Weed. System. Zool. 4: 75-82. --, 1956 Mutant color patterns in leopard frogs. A possible multiple allelic series. J. Heredity 47: 79-85. - , 1957 Genetic aspects of anuran populations. Am. Naturalist 91: 355-372. -, 1960 Interaction of mutant genes in the leopard frog. J. Heredity 51: 151-155. -, 1961 Variable expressivity of a mutant gene in the leopard frog. Science 134: 102-104. -, 1963 Interplay of mutant and wild-type pigment cells in chimeric leopard frogs. Devel. Biol. 8: 205-221. - , 1964 Fate of

The genetics of the Tettigidae. V. Bibliog. Genet. 5: 27-104.

Anat. Record 78: 139-147.

VOLPE, E. P., 1955


neural crest homotransplants in pattern mutants of the leopard frog. J. Exptl. Zool. 157: 179-196. -, 1970 Chromosome mapping in the leopard frog. Genetics 64: 11-21.

VOLPE, E. P. and S. DASGUPTA, 1962 Effects of different doses and combinations of spotting genes in the leopard frog R a m pipiem. Devel. Biol. 5 : 264-295.

WEED, A. C., 1922 ZWEIFEL, R. G., 1968

New frogs from Minnesota. Proc. Biol. Soc. Washington 34: 107-110. Reproductive biology of Anurans of the arid Southwest, with emphasis

on adaptation of embryos to temperature. Bull. Am. Mus. Nat. Hist. 140: 1-64.

APPENDIX A

AGE AT SEXUAL MATURITY

It has been reported that northern Ram pipiens ordinarily require two years or more to reach sexual maturity in nature (FORCE 1933; RYAN 1953; MERRELL 1965). Eleven of the frogs shown in Table 9 were reared to sexual maturity, which is normally achieved at about 55 mm. snout- vent length. These frogs were fed Drosophila, mealworms, and crickets throughout the winter, and all surviving frogs exceeded 55 mm. in length in less than 15 months. Hand-rearing Rana pipiens to sexual maturity is rather difficult (VOLPE 1957; MCKINNELL 1962). These results indicate that even under the relatively unfavorable conditions in the laboratory, sexual maturity can be achieved in considerably less than two years (see also BROWDER 1967). Moreover, in one population under favorable conditions at a time of low population density in the breeding ponds, the tadpoles metamorphosed into frogs of about 50 mm. snout-vent length. In this population, which was being studied by mark-recapture methods, these young frogs grew to sexual maturity that same summer and bred the following spring when they were only one year old. This early maturation seemingly represents an adaptive response to low population density in natural populations.

APPENDIX B

RELATION BETWEEN LENGTH AND VOLUME IN TADPOLES

A recent request from a colleague in biochemistry who had searched the literature in vain for information on length-weight relations in tadpoles in order to calculate dosages leads to the inclusion of this note.

Because total length alone seemed to provide a rather inadequate measure of the magnitude of the size difference between tadpoles, the volumes of four tadpoles of different lengths were measured by water displacement. The results are shown in Table 12 and Figure 4. A plot of length and volume on semi-logarithmic paper produced a straight line. Since the specific gravity of the tadpole is very close to that of water, these figures also provide a rough approximation of the increase in weight of the tadpoles as they increase in length.

The growth curve that best expresses the relationship between the increase in length and the increase in volume appears to be the exponential function, y=ab" with a=0.01.8 and b=1.064, and with z as the length and y the volume (see PASTERNACH and GIANUTSOS, 1969). This par-

TABLE I2

Relation between length and volume of Rana pipiens tadpoles

Total length Volume displaced Tadpole (mm) (ml)

23 46 70 85

0.2 1 .o 3.7 8.1

PIGMENT PATTERNS I N FROGS 161

ticular curve is most useful when z increases linearly with time but the increase in y is expontial. It gave a better fit to the data than the power function y=azb where both z and y increase exponentially with time.

0- LENGTH (mm)

FIGURE 4.-Relationship between length in mm and volume in ml of four tadpoles of R a m pipiens.

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LABORATORY STUDIES BEARING ON PIGMENT ... - genetics.org · LABORATORY STUDIES BEARING ON PIGMENT PATTERN POLYMORPHISMS IN WILD POPULATIONS OF RANA PIPIENS DAVID J. MERRELL Department