A Test of the Trivers-Willard Hypothesis on a

Monogamous Colonial Bird, The Black Tern

Bryanna Jacque
Madeline Kramer
Morgan Thomalla


Loras College Department of Biology
1450 Alta Vista St.
Dubuque, Iowa 52001


Abstract
The purpose of our experiment is to test the Trivers-Willard Hypothesis on female Black Terns
and their offspring. The Trivers-Willard Hypothesis states that female parents can control the
sex of their offspring based on their offsprings future mating success, despite the fact that sex
determination is chromosome determined. The Trivers-Willard Hypothesis is generally applied
to polygnous animals, but we intend to apply it to black terns, which as far as we know, are
monogamous animals. We will be extracting DNA from the blood samples of black tern
offspring and analyzing it to determine the sex of the birds. We will then compare the sex-ratio
of the offspring with the quality of the mothers to see if there is any connection between good
quality females and the production of sons. If this hypothesis can be supported, one could
assume that both polygnous and monogamous animals can control the sex of their offspring.
However, we found that the quality of the female does not have a significant effect on the sex of
her offspring. Consequently, our results support the idea that the Trivers-Willard Hypothesis
only applies to polygnous animals, and allows us to reject our own hypothesis.

Introduction
The Trivers-Willard Hypothesis predicts that, under certain circumstances, low-quality
mothers will produce daughters whereas high-quality mothers will produce sons (Wild & West,
2007). The reasoning behind this is that if low-quality mothers produce sons, they are going to
be less likely to mate because they are going to be weaker coming from a low-quality mother.
Daughters, on the other hand, will be able to find a mate regardless of their quality (Wild &
West, 2007). A high quality female should produce sons because the sons have a higher mating
success because they will also be at a higher quality.
The Trivers-Willard hypothesis is generally applied to polygynous mammals. It has been
reported in some mammals that healthy or high-quality mothers tend to produce more sons, and
unhealthy mothers produce more daughters (Frank, 1990). In polygnous birds, male offspring
tend to be larger in size than female offspring, therefore it would be less likely for a low-quality
mother to produce a large, strong male. Since daughters are smaller in size and generally have at
least some mating success no matter what their quality is, low-quality birds are more likely to
produce daughters (Ramsay, et al., 2003). The Trivers-Willard Hypothesis is also seen in small
polygnous mammals. In a study on small polygnous mammals, females were more likely to
carry a litter of small female pups, rather than a litter of large male pups because it was less
strenuous to carry small females. In doing so, the mother would be able to produce more food
for her offspring since it does not require as much energy to carry females compared to males
(Koskela, et al., 2009).
However, this is different for monogamous birds because they are all the same size,
therefore have equal mating success. In monogamous sea birds for example, well-fed parents
will produce males because they are stronger and therefore more fit to produce the larger male
offspring (Merkling, et al., 2012).
Black terns are monogamous colonial birds that tend to be relatively equal in size to each
other. Our objective is to see if the Trivers-Willard hypothesis applies to black terns. Low-
quality mothers would most likely still produce low-quality sons with minimal mating success;
therefore, low-quality mothers would produce daughters instead. We propose to run DNA
analyses on the blood samples of black tern offspring to determine the sex of the offspring.
After we have finished running our samples, we will create an index of what is seen as a high-
quality and what is seen as a low-quality mother. Then we will be given the information on
which offspring belong to which parents. We will then see if there is any sign that the Trivers-
Willard hypothesis does in fact apply to black terns using the results from our statistical analysis.

Methods
Step One: The autoclaved tubes were labeled with the birds identification numbers and then the
blood samples were vortexed. 100 L of black tern offspring blood, pre-obtained by Dr. Shealer
from Black Tern colony at Horizon Marsh in Dodge County, Wisconsin, was pipetted into a new
autoclaved tube. 100 L of T
10
E
1
was added to the blood samples followed by 2 L of 10 mg/ml
stock Proteinase K. The samples were vortexed for about 10 seconds or until mixed well. The
samples were microfuged for 20 seconds to bring the contents to the bottom. Finally the samples
were incubated at 55 C for 1 hour or more.
Step Two: 200 L of Tris-saturated phenol was added to the samples for the first DNA
extraction. The samples were then vortexed for 10 to 20 seconds and spun in the microfuge for 7
minutes at top speed. The top aqueous layer was removed with a pipet into a new labeled
autoclaved tube. We added 200 L T
10
E
1
saturated phenol: chloroform: isoamyl alcohol 25:24:1
to the sample next. The samples were vortexed for 20 to 30 seconds and then microfuged them
on high speed for 7 minutes. Again, the top aqueous layer was removed and added to a fresh,
labeled, autoclaved tube. The samples were vortexed for 20 to 30 seconds and then microfuged
on high speed for 7 minutes. 20 L of 3M Sodium acetate and 500 L of 100% ethanol are
added, the samples are inverted. The samples were placed the freezer that was set at -80 C for
one hour or more in order to precipitate the DNA.
Step Three: The samples were microfuged for 10 minutes at full speed. A pipet was used to
remove the ethanol from the DNA pellet. The DNA pellet was washed with 200 L of 70%
ethanol and inverted gently. Samples were spun in the microfuge for 2 minutes at full speed.
The ethanol from DNA pellet was removed and rewashed with 200 L of 70% ethanol. Ethanol
was removed as much as possible and the DNA pellets were left to air dry.
Step Four: The DNA pellet was suspended in 20-50 L T
10
E
1 .
and left to sit for 24 hours or
more, so the pellets could dissolve completely.
Step Five: A gel of 0.8g agarose, 100 ml of buffer, and 3 L ethidium bromide was prepared. 2
L of dye and 10 L of DNA were mixed together and then loaded into the gel comb slots. The
gel was run for 45 minutes at 150 volts in 1X TAE buffer. The completed gel was placed under
a black light and the DNA was categorized by the level of DNA present.
Step Six: A master mix of 44 L of 10X PCR Buffer, 35.2 L of 25mM MgCl
2
, 8.8 L of 10
mM dNTP mix, 8.8 L of 50 M primer #1, 8.8 L of 50 M primer #2, and 2.2 L of 5 l of
Taq Polymerase was made for ten samples and one negative control. Enough H
2
O and DNA
were added to each PCR tube to equal a total of 5 L. Taq polymerase was added to the master
mix and vortexed briefly. 35 L of master mix was then dispensed into the PCR tubes, capped,
and vortexed gently. The tubes were thermocycled for 5 minutes at 94
o
C followed by 40 cycles
of 94
o
C for 1 minute, 48
o
C for 1 minute, 72
o
C for 1 minute. A 1.8% agarose gel was prepared
in 1X TAE buffer and ran at 150 volts for 70 minutes. Sex of the offspring was determined by
viewing the gel under a black light.
List of materials needed:
-Autoclaved Tube
-Pipets
-Pipet Tips
-Blood Samples
-Vortex
-Microfuge
-Incubator set at 55 C
-T
10
E
1
saturated phenol: chloroform: isoamyl alcohol 25:24:1
-Proteinase K
-Tris-saturated Phenol
-3M Sodium Acetate
-100% Ethanol
-70% Ethanol
-Freezer set at -80 C
Results:
The mean of the BC Index was 0.30413 and the median was 0.29900. The mean number
of male chicks was 1.65 and the median was 2.0.

Figure 1:

Figure 1 shows the distribution of the body sizes measured by body mass divided by head length,
cubed.








Figure 2:

Figure 2 shows the distribution of the number of male chicks per nest. It shows the number of
male chicks per nest is 1.65.







Table 1:

Model Model
Fitting
Criteria Likelihood Ratio Tests
-2 Log
Likeliho
od
Chi-
Square df Sig.
Intercept
Only
64.567

Final 62.238 2.330 3 .507
Table 1 explains how well the model fits the data, in comparison to the model with only the
intercept. It also tells us that the female body did not have a significant effect on sex ratio which
was seen by the p-value of 0.507.

Table 2:
Pseudo R-Square
Cox and Snell .072
Nagelkerke .079
McFadden .031
Table 2 shows that 72% of the data was explained by the model.
Table 3:

Parameter Estimates
NumMale
a

B Std. Error Wald df Sig. Exp(B)
95% Confidence Interval for
Exp(B)
Lower Bound Upper Bound
0 Intercept -19.675 13.553 2.107 1 .147

BCIndex 60.434 43.616 1.920 1 .166 1.763E26 1.319E-11 2.357E63
1 Intercept -8.044 9.657 .694 1 .405

BCIndex 29.247 32.199 .825 1 .364 5.033E12 1.969E-15 1.287E40
2 Intercept -4.819 10.077 .229 1 .633

BCIndex 17.776 33.653 .279 1 .597 5.250E7 1.187E-21 2.322E36
Table 3 shows that each female Black Tern that had 0 male offspring, each that had 1 offspring, and each that had
2 offspring, compared to those that had 3 offspring. The slope of 0 male offspring was 60.434, 29.247 for 1 male,
and 17.776 for 2 male offspring.

Discussion
Our hypothesis was that high quality female Black Tern would produce more sons. Our
p-vaule of 0.507 shown in Table 1 does not support our hypothesis that high quality birds will
produce sons and low quality birds will produce daughters. The p-value tells us that the quality
of the females did not have a significant effect on the sex ratio of their offspring. Table 2 tells us
that 72% of our data fits the model.
Figure 1 shows the distribution of body masses for the female black terns. It told us that
the mean of the BC Index was 0.30. These distributions were based on the quality of the mother
as mass divided by head length cubed. Birds with a smaller head length and heavier weight were
considered higher quality females than those with a larger head length and lighter weight. Figure
2 shows us the distribution of the number of sons, out of three broods, that each female
produced. The average numbers of sons in a nest of three was 1.65 sons. The number of sons
that occurred most often was one son per nest, while the number of sons that occurred least often
was zero sons per nest.
Table 3 tells us that none of the male chicks per nest were significant when comparing it
to the BC Index, so the quality of the female did not have a significant effect on the sex ratio of
sons produced. (We need to go over this better with you. We are confused about how to
interpret it.)
The other studies we looked at mostly focused on the effect of the Trivers-Willard
Hypothesis on polygnous animals. Most of these studies supported the Trivers-Willard
Hypothesis. We did find a study on monogamous sea-birds, however, which supported the idea
that well-fed parents were more likely to produce sons (Merkling, et al., 2012). Although male
and female Black Terns are about equal in size, unlike the rest of the animals that were in the
studies we researched, we thought that the Trivers-Willard Hypothesis would still apply to them
since it seemed to apply to many polygnous animals and even the monogamous sea-birds.
Our results were able to help support the idea that the Trivers-Willard Hypothesis does
not apply to monogamous birds. However, we had a fairly small sample size for our data, so
having a larger sample size might have changed the results. In the future, if this study is
repeated, a larger sample size should be used. To continue to test the effect of the Trivers-
Willard Hypothesis on monogamous birds, different species of birds should be tested.


Literature Cited
Frank, S. A. (1990). Sex allocation theory for birds and mammals. Annual Review of
Ecology and Systematics, 21, 13-55.
Koskela, E., Mappes, T., Niskanen, T., & Rutkowska, J. (2009). Maternal investment in
relation to sex ratio and offspring number in a small mammal-a case for Trivers
and Willard theory. Journal of Animal Ecology, 78(5), 1007-1014.
Merkling, T., Leclaire, S., Danchin, E., Lhuillier, E., Wagner, R. H., White, J., Hatch
S.A., & Blanchard, P. (2012). Food availability and offspring sex in a
monogamous seabird: insights from an experimental approach. Behavioral
Ecology, 23(4), 751-758.
Ramsay, S. M., Mennill, D. J., Otter, K. A., Ratcliffe, L. M., & Boag, P. T. (2003). Sex
allocation in black-capped chickadees. Journal of Avian Biology, 34, 134-139.
Wild, G., & West, S. A. (2007). A sex allocation theory for vertebrates: Combining local
resource competition and conditiondependent allocation. The American
Naturalist, 170(05), 112-128. doi: 10.1086/522057