Download Rate of molecular evolution of the seminal protein gene

© 2004 Nature Publishing Group http://www.nature.com/naturegenetics
LETTERS
Rate of molecular evolution of the seminal protein gene
SEMG2 correlates with levels of female promiscuity
Steve Dorus1,2, Patrick D Evans1,2, Gerald J Wyckoff1,3, Sun Shim Choi1 & Bruce T Lahn1
Postcopulatory sperm competition is a key aspect of sexual
selection and is believed to drive the rapid evolution of both
reproductive physiology and reproduction-related genes1–4. It is
well-established that mating behavior determines the intensity
of sperm competition, with polyandry (i.e., female promiscuity)
leading to fiercer sperm competition than monandry1–3. Studies
in mammals, particularly primates, showed that, owing to
greater sperm competition, polyandrous taxa generally have
physiological traits that make them better adapted for
fertilization than monandrous species, including bigger testes,
larger seminal vesicles, higher sperm counts, richer
mitochondrial loading in sperm and more prominent semen
coagulation2,5–8. Here, we show that the degree of polyandry
can also impact the dynamics of molecular evolution.
Specifically, we show that the evolution of SEMG2, the gene
encoding semenogelin II, a main structural component of
semen coagulum, is accelerated in polyandrous primates
relative to monandrous primates. Our study showcases the
intimate relationship between sexual selection and the
molecular evolution of reproductive genes.
competition7. Given the strong influence of the primate mating system
on the intensity of sperm competition, we sought to examine whether
the molecular evolution of semenogelin in primates is subject to
different selective regimes under different mating systems.
We obtained the coding sequence of SEMG2 from a diverse
phylogenetic range of primates: five great apes (human, Homo sapiens;
common chimpanzee, Pan troglodytes; pigmy chimpanzee, Pan
paniscus; gorilla, Gorilla gorilla; and orangutan, Pongo pygmaeus),
two lesser apes (white-handed gibbon, Hylobates lar; and Kloss
gibbon, Hylobates klossii), three macaque monkeys (rhesus macaque,
Macaca mulatta; crab-eating macaque, Macaca fascicularis; and
pig-tailed macaque, Macaca nemestrina), one colobus monkey
(black-and-white colobus, Colobus guereza) and one New World
monkey (spider monkey, Ateles geoffroyi). We chose these species
0.91
1.27
∞
∞
0.67
Orangutan
1.31
Catarrhine
ancestor
0.83
∞
0.86
1.39
Kloss gibbon
Lesser apes
Pig-tailed macaque
∞
0.70
Colobus monkey
1.86
30
20
Million years ago
White-handed gibbon
Crab-eating macaque Old World
1.73 0.82
monkeys
Rhesus macaque
7.05
40
Pygmy chimpanzee
Common chimpanzee Great apes
Gorilla
0.88
0.58
In primates, semenogelin is a main protein constituent of the seminal
fluid produced by seminal vesicles. After ejaculation, semenogelin
undergoes covalent cross-linking to become the principal structural
component of semen coagulum in the reproductive tract of recipient
females. Over time, the coagulum is liquefied through the proteolytic
cleavage of semenogelin by the prostate-derived protease kallikrein 3
(also known as prostate-specific antigen), a process that leads to the
release of sperm from the coagulum. It has been argued that this finely
orchestrated process of postcopulatory semen coagulation and subsequent dissolution of the coagulum is crucial in preventing fertilization of a recently inseminated female by rival males in subsequent
copulations9,10. Alternative mechanisms to this ‘chastity belt’ model
have also been proposed11,12, including the prevention of sperm loss
from backflow13,14 and the protection of spermatozoa during their
progression through the female reproductive tract6. Regardless of the
actual mechanism by which semen coagulation promotes fertilization,
the coagulation process probably has an important role in sperm
Human
1.02
3.15
Spider monkey
10
New World
monkey
0
Figure 1 Lineage-specific o values of SEMG2 in primates, calculated
using the SEMG2 coding region before the stop codon present in the
chimpanzees, which occurs earlier than in the other primates17. The
phylogeny is drawn roughly to scale of evolutionary time. N, dN is nonzero
and dS is zero. The o value of 1.86 shown next to the spider monkey branch
applies to the entire lineage from the catarrhine ancestor node (indicated
by arrow) to spider monkey.
1Howard
Hughes Medical Institute, Department of Human Genetics; and 2Committee on Genetics, University of Chicago, Chicago, Illinois 60637, USA.
address: Division of Biology and Biochemistry, University of Missouri-Kansas City, Kansas City, Missouri, 64108, USA. Correspondence should be
addressed to B.T.L. ([email protected]).
3Present
Published online 7 November 2004; doi:10.1038/ng1471
1326
VOLUME 36
[
NUMBER 12
[
DECEMBER 2004 NATURE GENETICS
LETTERS
tree, o values are much greater than 1, which is suggestive of positive
selection. The codon-based maximum likelihood test19 confirmed
statistically that the pattern of SEMG2 sequence evolution in primates
can be significantly better explained by the evolutionary model that
invokes positive selection than by models that lack positive selection
(P 5 0.0001; Supplementary Tables 1 and 2 online). Furthermore,
the test showed a very high o value (6.70) on the codon sites identified
to be positively selected (Supplementary Table 1 online).
Having shown that the overall pattern of SEMG2 evolution in
primates is consistent with positive selection, we next sought to
examine the effect of selection in individual primate lineages. Specifically, we wished to investigate whether there is a correlation between
the rates of SEMG2 evolution and the previously reported levels of
polyandry in different primate species6,20–25. For human, gorilla,
orangutan and colobus monkey, we used the o value of the terminal
branch to estimate the rate of SEMG2 evolution. For the two highly
related chimpanzee species, each terminal branch was too short to
produce a reliable o value. We therefore combined the two terminal
chimpanzee branches with the adjacent internal branch leading to the
common ancestors of the two chimpanzees to produce a single, more
reliable o value for the chimpanzee genus. For the same reason, we
combined the branches corresponding to the three highly related
macaque species along with the adjacent internal branch leading to
these macaques to produce a single o for the macaque genus. Finally,
we combined the two gibbon branches and the adjacent internal
branch to produce a single o for the gibbons. We omitted the spider
monkey in the comparison because the lineage leading to it is too long
to represent the recent evolution of the species. As shown in Table 1,
there is a trend for species with greater levels of polyandry to have
higher o values. Plotting o against the average number of male
partners per periovulatory period of the female showed a robust
positive correlation (Fig. 2a; P o 0.0001). Furthermore, although
spider monkey was excluded from the above analysis, its o value is
rather high, which is consistent with the polyandrous mating behavior
of this species (spider monkey uses the promiscuous multimalemultifemale mating system, where a female averages several male
partners per periovulatory period6,26).
The level of polyandry substantially affects the reproductive physiology of primates2,5–8. Polyandrous species have larger testes and
more prominent semen coagulation. We therefore sought to investigate whether the rate of SEMG2 evolution in primates is correlated
with testis size. We plotted o values of the various primates against
their residual testis size as previously described8 and found a positive
Table 1 Rates of SEMG2 evolution and levels of polyandry in
primates
Mean number
Mating system
of male partners per
periovulatory period
o
Multimale-multifemale
Multimale-multifemale
B8
B3
2.52
1.28
Various
Dispersed
1–2
1–2
0.91
0.88
Monogamous
Polygynous
B1
B1
0.89
0.70
Polygynous
B1
0.61
Species
© 2004 Nature Publishing Group http://www.nature.com/naturegenetics
Chimpanzees
Macaques
Humana
Orangutanb
Gibbons
Colobus monkey
Gorilla
aMating
systems in humans are varied and continuously changing due to cultural influence. The
mean number of partners is probably somewhat greater than one, though the precise mean
across culture and time is difficult to assess. bOrangutans are solitary, making it difficult to
assess accurately the number of male partners. But given documented opportunistic mating
with nonterritorial males in addition to territorial males, the mean number of male partners per
periovulatory period is thought to be somewhat greater than one.
because they represent the main mating systems in primates: monogamy, polygyny, multimale-multifemale and dispersed6,15. The first two
mating systems are believed to correspond to monandry, defined as
one female copulating with one male during each periovulatory period.
The third mating system features high levels of polyandry, where one
female copulates with multiple males in a single periovulatory period.
The last mating system, which is found only in orangutan among the
species sampled here, is believed to fall somewhere in between
monandry and polyandry. In orangutans, a female copulates with
the territorially dominant male during a given periovulatory period
but can also copulate with additional nonterritorial males through
opportunistic mating6,16. Our sampling is intended to represent diverse
primate mating systems and is particularly focused on apes and Old
World monkeys, where social structure and reproductive behavior have
been well studied and are particularly diverse among species.
We constructed a phylogenetic tree from SEMG2 sequences of these
species using the SEMG2 coding region from the start codon present
in all primates to the stop codon present in the chimpanzees, which is
earlier than in the other primates17. We obtained the nonsynonymous
substitution rate (dN) and the synonymous substitution rate (dS) for
each branch of the tree using the free-ratio maximum likelihood
method18. We then calculated the dN/dS ratio (called o), which
measures the rate of protein evolution as scaled to mutation rate,
for all the branches (Fig. 1). We noted that, for many lineages of the
a
4.0
b
3.5
r 2 = 0.98
Chimpanzees
0.5
0
Macaques
Gibbons
2
3
2.5
2.5
2.0
2.0
1.5
1.5
1.0
Human
Orangutan
Gorilla
Colobus monkey
1
0.5
4
5
6
7
3.0
Chimpanzees
2.0
1.0
r 2 = 0.52
3.0
2.5
Gibbons
Gorilla
0
–2.0
8
4.0
3.5
3.5
3.0
1.5
c
4.0
Orangutan
Macaques
Human
Colobus monkey
1.0
Chimpanzees
Human
Gibbons
Gorilla
Macaques
Orangutan
Colobus monkey
0.5
0
–1.5
–1.0
Mean number of male partners per periovulatory period
–0.5
0
Residual testis size
0.5
1.0
1.5
2
3
4
Semen coagulation rating
Figure 2 Correlation between the rate of evolution of SEMG2 and reproductive behavior and physiology in primates. The o values are calculated using
the SEMG2 coding region before the stop codon present in the chimpanzees, which occurs earlier than in the other primates17. (a) Correlation between
species- or genus-specific o and mean number of male partners per periovulatory period of the female. (b) Correlation between species- or genus-specific
o and residual testis size. Residual testis sizes of primates are as previously described8. (c) Relationship between species- or genus-specific o and the
extent of semen coagulation. Semen coagulation ratings of primates are as previously described7.
NATURE GENETICS VOLUME 36
[
NUMBER 12
[
DECEMBER 2004
1327
© 2004 Nature Publishing Group http://www.nature.com/naturegenetics
LETTERS
correlation between these two measurements (Fig. 2b; P ¼ 0.035).
Next, we examined the relationship between o and the extent of
semen coagulation. Using the Dixson and Anderson rating of semen
coagulation7, we found a trend for species with higher semen
coagulation ratings to have higher o values (Fig. 2c). Given that
semenogelin forms the main constituent of semen coagulum through
chemical cross-linking, this result suggests that the molecular evolution of SEMG2 may directly influence the biochemical dynamics of
semen coagulation.
Our analysis was done on terminal lineages because they are the best
available proxies for the recent evolution of the species. A caveat of
such analysis is that the mating system of an extant species may not be
the same as its earlier progenitors in the lineage. This caveat is mollified
by two considerations. First, we deliberately focused on short lineages
and excluded long lineages, such that the current mating system of a
species is more likely to be representative of the lineage. Second, our
approach, though imperfect, makes our conclusion more conservative.
As an example, consider two sister species with different mating
systems. Their reproductive systems were initially nearly identical at
the point of speciation but gradually differentiated from each other as
they diverged over evolutionary time. As such, the o value of one of
these lineages represents the composite impact of selection from when
the mating systems were very similar between the two lineages, just
after speciation, and when the mating systems became very different
later. As such, our analysis is an underestimate of differential, speciesspecific selection associated with the evolution of distinct mating
systems. Despite this underestimation, there is a pattern in the primate
phylogeny of SEMG2, whereby the rate of evolution correlates tightly
with both the level of polyandry and reproductive physiologies such as
testis size and semen coagulation rating.
As mentioned earlier, the analyses described thus far were done on
the portion of the SEMG2 coding region up to the chimpanzeespecific stop codon, which is earlier than the stop codon in the other
primates17. This is a stringent approach because only the portion of
the gene that is functional in all the species is used for sequence
comparisons. But this approach ignores a large portion of the gene
(B30%) that is probably functional in most species. We therefore
decided to complement the above studies by repeating the analyses
with the entire coding region of the gene. We felt that this strategy,
though imperfect, would be conservative for the following reason.
When considering only the coding region up to the chimpanzeespecific stop codon, the o value of the chimpanzee genus is much
greater than 1. The addition in the chimpanzees of the region after the
stop codon would, in theory, tend to negate signatures of positive
selection (such as o greater than 1), given that this region has
presumably evolved neutrally in the terminal chimpanzee branches.
Analyses using the entire coding region showed the same trends
(Supplementary Figs. 1 and 2 online), which reinforces the robustness of these trends.
To identify the domains of SEMG2 most affected by selection, we
used a sliding-window analysis of o on the two lineages that showed
the greatest acceleration of protein evolution: the common chimpanzee lineage after human-chimpanzee divergence, and the lineage from
the catarrhine ancestor to crab-eating macaque. For these two lineages,
o is substantially greater than 1 for most of the gene and is especially
high in the upstream portion. In particular, o approaches the
unusually high value of 14 around the domain associated with
sperm motility inhibition27 (Fig. 3). Furthermore, among the codons
identified by the codon-based maximum likelihood analysis19 to have
experienced positive selection, all but two are located in the upstream
half of the protein (Supplementary Table 1 online). These results
1328
Exon 1
Exon 2
Sperm motility–inhibition domain
60-aa repeat domain
16
Human/chimpanzee ancestor to common chimpanzee
Catarrhine ancestor to crab-eating macaque
14
12
10
8
6
4
2
0
0
200
400
600
800
1,000
1,200
1,400
1,600 1,800
Position of window center (bp)
Figure 3 Sliding-window analysis of o values in the common chimpanzee
lineage and the crab-eating macaque lineage. Dotted line indicates o of 1.
Values of o far exceed 1 in most regions of the gene, particularly around the
sperm motility–inhibition domain.
suggest that the timing and the extent of the biochemical inhibition of
sperm motility in the coagulum may be a key target of selection.
We also examined the molecular evolution of KLK3, encoding
kallikrein 3, which liquefies semen coagulum by cleaving semenogelin28. We found no evidence of positive selection operating on KLK3.
The average pairwise o for KLK3 is 0.29, much lower than the value
for SEMG2. This suggests that the process of coagulum liquefaction
is under weak or no positive selection as compared with the process
of coagulum formation.
It would be of great interest to know what biochemical properties of
SEMG2 are under positive selection. This knowledge awaits future
studies that specifically address how sequence evolution in SEMG2
alters its biochemical properties.
As first recognized by Charles Darwin in The Descent of Man, and
Selection in Relation to Sex, sexual selection is a powerful force in the
evolution of species. An extensive body of work addresses the impact
of sexual selection on various phenotypic traits, especially those
relating to reproductive physiology. More recently, studies are beginning to address the influence of sexual selection on the molecular
evolution of genes1,3,4. To our knowledge, the current study represents
the first demonstration that the molecular evolution of a reproductive
gene is differentially influenced by different levels of sexual selection
across diverse species. As such, the study showcases the intimate
relationship between sexual selection and the molecular evolution of
reproductive genes.
METHODS
DNA sequencing and analysis. We obtained SEMG2 sequences experimentally
from the common chimpanzee, lowland gorilla, orangutan, white-handed
gibbon, crab-eating macaque, pig-tailed macaque, black-and-white colobus
monkey and spider monkey. We obtained KLK3 sequences experimentally from
the common chimpanzee, lowland gorilla and orangutan. For these species, we
obtained PCR products from genomic DNA and then sequenced PCR
products. We obtained additional SEMG2 and KLK3 sequences from GenBank
for human (SEMG2 and KLK3) and for pigmy chimpanzee, Kloss gibbon and
rhesus macaque (SEMG2). For SEMG2, we calculated lineage-specific dN and
dS values using the free-ratio maximum likelihood method18. For KLK3, we
obtained pairwise dN and dS values using the Yang and Nielson method29.
Evolutionary and statistical analysis. We assessed evidence of positive selection in the primate phylogenetic tree of SEMG2 by the codon-based maximum
VOLUME 36
[
NUMBER 12
[
DECEMBER 2004 NATURE GENETICS
© 2004 Nature Publishing Group http://www.nature.com/naturegenetics
LETTERS
likelihood analysis19 using the Codeml program in the PAML package. For
technical details of this test, please refer to the original description19. We
compared the likelihood fit of three evolutionary models: one-ratio (M0),
neutral (M1) and positive selection (M2). The program produced the number
of parameters used in the analysis, the o values and the log likelihood of each
model. With these parameters, we calculated the probability that two models
should differ in log likelihood as much as observed, given the degree of
freedom, using the Akaike Information Criterion as described19. If the model
allowed positive selection, the program also indicated all the sites that have
probably experienced positive selection19. Results of the Likelihood Ratio
analysis are given in Supplementary Tables 1 and 2 online. For sliding-window
analysis of o values, we calculated the nonsynonymous and synonymous
substitution rates using the Li method30 as implemented by the Wisconsin
Package v10.2 (Accelrys), with nonsynonymous rate calculated for window size
of 100 codons and sliding increment of one codon, and synonymous rate of the
entire gene used as denominator to avoid problems associated with stochastic
variation that can lead to division by zero. We calculated linear regression and
r2 value of correlation in Microsoft Excel and determined the statistical
significance of correlation by the standard t-statistic.
URL. The PAML package is available at http://abacus.gene.ucl.ac.uk/software/
paml.html.
GenBank accession numbers. SEMG2 sequences: human, NM_003008; common chimpanzee, AY781386; pigmy chimpanzee, AY259288; lowland gorilla,
AY781387; orangutan, AY781388; white-handed gibbon, AY781389; Kloss
gibbon, AY259291; rhesus macaque, X92589; crab-eating macaque,
AY781390; pig-tailed macaque, AY781391; black-and-white colobus monkey,
AY781392; spider monkey, AY781393. KLK3 sequences: human, NM_001648;
common chimpanzee, AY781394; lowland gorilla, AY781395; orangutan,
AY781396.
Note: Supplementary information is available on the Nature Genetics website.
ACKNOWLEDGMENTS
We thank W.-H. Huang for technical assistance; A. Di Rienzo, C. Malcom,
N. M. Pearson, E. J. Vallender and C.-I Wu for discussions and comments on the
manuscript; and L.G. Chemnick, A.R. Ryder and L. Faust for providing primate
tissue samples. This work was supported in part by the William Rainey Harper
Fellowship (to S.D.) and the Searle Scholarship and the Burroughs Wellcome
Career Award (to B.T.L.).
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Received 25 June; accepted 15 October 2004
Published online at http://www.nature.com/naturegenetics/
1. Karr, T.L. & Pitnick, S. Sperm competition: defining the rules of engagement.
Curr. Biol. 9, R787–R790 (1999).
2. Dixson, A. & Anderson, M. Sexual selection and the comparative anatomy of reproduction in monkeys, apes, and human beings. Annu. Rev. Sex Res. 12, 121–144 (2001).
NATURE GENETICS VOLUME 36
[
NUMBER 12
[
DECEMBER 2004
3. Birkhead, T.R. & Pizzari, T. Postcopulatory sexual selection. Nat. Rev. Genet. 3, 262–
273 (2002).
4. Swanson, W.J. & Vacquier, V.D. The rapid evolution of reproductive proteins. Nat. Rev.
Genet. 3, 137–144 (2002).
5. Harcourt, A.H., Harvey, P.H., Larson, S.G. & Short, R.V. Testis weight, body weight and
breeding system in primates. Nature 293, 55–57 (1981).
6. Dixson, A.F. Primate Sexuality: Comparative Studies of the Prosimians, Monkeys, Apes,
and Human Being (Oxford University Press, New York, 1998).
7. Dixson, A.L. & Anderson, M.J. Sexual selection, seminal coagulation and copulatory
plug formation in primates. Folia Primatol. (Basel) 73, 63–69 (2002).
8. Anderson, M.J. & Dixson, A.F. Sperm competition: motility and the midpiece in
primates. Nature 416, 496 (2002).
9. Voss, R. Male accessory glands and the evolution of copulatory plugs in rodents. Occ.
Pap. Mus. Zool. Univ. Mich. 689, 1–17 (1979).
10. Kingan, S.B., Tatar, M. & Rand, D.M. Reduced polymorphism in the chimpanzee
semen coagulating protein, semenogelin I. J. Mol. Evol. 57, 159–169 (2003).
11. Dewsbury, D.A. A test of the role of copulatory plugs in sperm competition in deer mice
(Peromyscus maniculatus). J. Mammal. 69, 854 (1988).
12. Michener, G.R. Copulatory plugs in Richardson’s ground squirrels. Can. J. Zool. 62,
267–270 (1984).
13. Blandau, R.J. On factors involved in sperm transport through the cervix uteri of the
albino rat. Am. J. Anat. 73, 253–272 (1945).
14. Mathews, M.K. & Adler, N.T. Systematic interrelationships of mating, vaginal
plug position, and sperm transport in the rat. Physiol. Behav. 20, 303–309
(1978).
15. Dunbar, R.I. & Dunbar, E.P. Contrasts in social structure among black-and-white
colobus monkey groups. Anim. Behav. 24, 84–92 (1976).
16. Rodman, P.S. & Mitani, J.C. Orang-utans: sexual dimorhism in a solitary species
in Primate Societies (eds. Smuts, B., Cheney, D., Seyfarth, R., Wrangham, R. &
Struhsaker, T.) 146–154 (University of Chicago Press, Chicago, 1987).
17. Jensen-Seaman, M.I. & Li, W.H. Evolution of the hominoid semenogelin genes, the
major proteins of ejaculated semen. J. Mol. Evol. 57, 261–270 (2003).
18. Yang, Z. Likelihood ratio tests for detecting positive selection and application to
primate lysozyme evolution. Mol. Biol. Evol. 15, 568–573 (1998).
19. Nielsen, R. & Yang, Z. Likelihood models for detecting positively selected amino
acid sites and applications to the HIV-1 envelope gene. Genetics 148, 929–936
(1998).
20. Conoway, C.H. & Koford, C.B. Estrous cycles and mating behavior in a free-ranging
band of rhesus monkey. J. Mammal. 45, 577–588 (1965).
21. Tokuda, K., Simms, R.C. & Jenson, J.D. Sexual behavior in a captive group of pigtail
macaque (Macaca nemestrina). Primates 9, 283–294 (1968).
22. Van Noordwijk, M.A. Sexual behavior of Sumatran long-tailed macaques (Macaca
fascicularis). Z. Tierpsychol. 70, 277–296 (1985).
23. Goodall, J. The Chimpanzees of Gombe: Patterns of Behavior (Harvard University
Press, Cambridge, 1986).
24. Hasegawa, T. & Hiraiwai-Hasegawa, M. Sperm Competition and mating behavior. in
The Chimpanzees of the Mahale Mountains: Sexual and Life History Strategies
(ed. Nishida, T.) 115–132 University of Tokyo Press, Tokyo, 1990.
25. Manson, J.H. Mating patterns, mate choice and birth season heterosexual relationships
in free-ranging rhesus macaques. Primates 35, 417–433 (1992).
26. Strier, K.B. Faces in the Forest, the Endangered Muriqui Monkeys of Brazil (Oxford
University Press, New York, 1992).
27. Robert, M. & Gagnon, C. Purification and characterization of the active precursor of
a human sperm motility inhibitor secreted by the seminal vesicles: identity with
semenogelin. Biol. Reprod. 55, 813–821 (1996).
28. Lilja, H., Oldbring, J., Rannevik, G. & Laurell, C.B. Seminal vesicle-secreted proteins
and their reactions during gelation and liquefaction of human semen. J. Clin. Invest.
80, 281–285 (1987).
29. Yang, Z. & Nielsen, R. Estimating synonymous and nonsynonymous substitution rates
under realistic evolutionary models. Mol. Biol. Evol. 17, 32–43 (2000).
30. Li, W.H. Unbiased estimation of the rates of synonymous and nonsynonymous
substitution. J. Mol. Evol. 36, 96–99 (1993).
1329