Using a phenotypic engineering approach, Ketterson, Nolan and colleagues asked whether suites of hormone-mediated traits could represent both an adaptive outcome of past selection and a constraint to future evolution. In an early series of studies, they manipulated plasma hormone levels, identified a suite of testosterone-sensitive traits, and assessed their interactive relationship to fitness (reviewed in Ketterson & Nolan 1992
; Reed et al. 2006
). Their subjects were free-living males of a songbird species, the dark-eyed junco (Junco hyemalis
). Half the males in the population were treated with testosterone implants (T-males) at a dose that mimicked prolonged exposure to peak breeding levels; the other half of the males (C-males) received empty implants, thus maintaining normal levels of testosterone. Results demonstrated that experimentally enhanced testosterone increased male mating effort, as measured by song, courtship behaviour, home range size and success at obtaining extra-pair fertilizations (Ketterson et al. 1992
; Chandler et al. 1994
; Enstrom et al. 1997
; Raouf et al. 1997
; Reed et al. 2006
), but that the increase came at a cost both to parental behaviour, as measured by nestling feeding rate and nest defence, and to self-maintenance, as measured by body mass, immune function and survival (Ketterson et al
; Cawthorn et al. 1998
; Schoech et al. 1998
; Casto et al. 2001
; Reed et al. 2006
). An implication is that testosterone may mediate an adaptive trade-off between these functions. When fitness was modelled as relative potential for population growth, T-males had higher fitness than C-males, because the benefits of enhanced mating success outweighed the costs of reduced survival and parental care (Reed et al. 2006
). The implication here is that seemingly fit possible phenotypes ‘located’ in the genome—those uncovered by experimental elevation of testosterone—are not normally expressed, and the ensuing question is why.
More recent research on juncos has addressed these implications. First, we asked whether natural variation in male testosterone does in fact underlie an adaptive resolution of the trade-off between mating effort and parental effort/survival at the level of individual variation. Second, we asked why natural counterparts to T-males are not more common in nature. Anticipating that selection on males might lead to correlated response in females, we asked whether male response to selection favouring expression of the full range of possible male phenotypes might be constrained by the detrimental impact that selection could have on females.
To address the first question, we assessed natural variation in male testosterone, focusing our attention on variation in undisturbed circulating levels and on male capacity to produce short-term testosterone increases. In many songbirds (including closely related sparrows), natural testosterone levels fluctuate rapidly in response to social stimuli such as competing males or potential mates (e.g. Wingfield 1985
; Pinxten et al. 2003
; Landys et al. 2007
). Social modulation of circulating testosterone, which is the foundation of the ‘challenge hypothesis’, is thought to allow males to produce testosterone only when circumstances call for it, lessening the costs that would accompany constitutively high testosterone levels (Wingfield et al
; Goymann et al. 2007
). To assess variation among male juncos in their baseline levels of testosterone and their capacity to produce short-term testosterone increases, we used injections of gonadotropin-releasing hormone (‘GnRH challenges’) to stimulate the hypothalamic-pituitary-gonadal (HPG) axis to produce transient increases of plasma testosterone. Importantly, when males were challenged multiple times across the breeding season, the magnitude of the short-term testosterone increase (i.e. the difference between post- and pre-challenge levels) was found to be repeatable (Jawor et al. 2006
). Although we have yet to measure heritability due to the difficulty of obtaining a large sample of relatives, individual consistency suggests the possibility of genetic variance underlying variation in short-term testosterone increases. GnRH challenge response is not only repeatable but also ecologically relevant, as the testosterone levels generated by GnRH challenges predicted levels produced in response to a male territorial intruder (McGlothlin et al. 2008
To determine whether natural variation in testosterone is related to the trade-off between mating effort and parental effort, as predicted by the implant studies, we again used GnRH challenges and correlated the results with the results of standardized protocols to elicit mating-related and parental behaviour. As a measure of mating effort, we assessed territorial aggression in response to simulated territorial intrusions (Wingfield 1985
). As a measure of parental effort, we assessed nestling feeding rate. With respect to aggression, we found that the peak testosterone levels produced in response to GnRH predicted a male's level of aggression toward the intruder, which indicates that males with more responsive HPG axes invest more effort in territorial defence (McGlothlin et al. 2007
). With respect to parental behaviour, we found that males with larger changes in testosterone levels in response to GnRH made fewer trips to the nest during nestling feeding (McGlothlin et al. 2007
). These correlations provide compelling evidence that natural variation in testosterone production affects a fundamental life-history trade-off, thus confirming expectations generated by studies based on experimental testosterone elevation.
Critically, however, if we are to know whether the suite of traits mediated by testosterone is the adaptive outcome of correlational selection on the traits comprising the suite, we will need to relate response to GnRH to different components of fitness. This effort is currently underway. Future studies should also address two caveats. First, although the primary function of territorial defence is to maintain a breeding location, making it a reasonable proxy for mating effort, future studies should measure behaviours such as courtship to determine whether they too are related to testosterone in response to GnRH. Second, these studies correlating behaviour to response to GnRH were not conducted on the same individuals at the same time. Future studies should confirm that testosterone in response to GnRH covaries with territorial aggression and parental behaviour when all are measured in the same individuals.
The results also raise the important question of why individual males should vary in the resolution of mating effort/parental effort trade-off, and thus in testosterone production, at all. Another study suggests a possible reason why some males should be submissive and parental while others are aggressive and less parental. The magnitude of a male's testosterone increase in response to GnRH was found to be positively correlated with the size of the white plumage patch on its tail (McGlothlin et al. 2008
). This patch, referred to as tail white, is displayed to females during courtship and other males during escalated aggressive encounters (Nolan et al. 2002
). Females prefer males with larger white patches (Hill et al. 1999
), and these males also tend to be socially dominant (Balph et al. 1979
; Holberton et al. 1989
). The correlation between tail white and testosterone, then, suggests that more attractive, dominant males tend to produce larger testosterone increases. Variation in the mating effort/parental effort trade-off may thus be linked to attractiveness.
Such an association could be maintained by correlational selection, which would arise if testosterone production and tail white interact in their effects on fitness. For example, it may be useless for a male to be attractive if he does not invest energy in the behaviours needed to obtain mates. Hence, mating behaviour and appearance are likely to interact in their effects on fitness, and attractive males that expend considerable effort may be highly successful at obtaining mates. As a correlate, however, unattractive males that spare investment in mating behaviour and focus on alternative routes to fitness may also benefit. Interactive fitness effects like this are likely to be common, and there may be many instances where correlational selection acts to associate mechanisms underlying trade-off resolution with attractive signals. Because there are several routes to obtaining fitness, this type of selection can also maintain variation in the resolution of trade-offs as well. As seen from this perspective, the suite of two behaviours and one plumage trait may act collectively as an adaptation allowing males to produce optimal levels of mating and parental effort depending on their attractiveness. If so, the absence of males similar to T-males may be explained by the frequency dependence of any advantage this phenotype might provide.
Up to this point, we have considered the evolution of testosterone-mediated suites from a solely male perspective. However, because males and females share much of their genome, male and female traits do not always follow completely independent evolutionary trajectories. Across species, mean testosterone levels of males and females tend to be correlated, suggesting that they may have coevolved (Wingfield 1994
; Ketterson et al. 2005
; Møller et al. 2005
; Mank 2007
). Within species, a genetic correlation across the sexes could act as a genetic constraint on the evolution of testosterone-mediated traits in males due to a correlated response to selection in females. To study this possibility, we examined the behavioural and fitness effects of experimentally elevated testosterone in females, and have begun preliminary studies of individual variation in females.
Studies of captive juncos have shown that females, like males, have decreased immune function when implanted with testosterone (Zysling et al. 2006
). Whereas downregulation of the immune system by testosterone may be adaptive for males (because it allows them to divert energy to mate acquisition), it may represent a net cost for females (Zuk 1990
). Also in captives, though not in free-living females, experimentally elevated testosterone inhibited brood patch formation (Clotfelter et al. 2004
). In the wild, testosterone implantation seemed to interfere with nest initiation, as time to first egg was longer in T-females (Clotfelter et al. 2004
). Incubation consistency and nest defence during the egg stage were unaffected (Clotfelter et al. 2004
). Another captive study suggested that testosterone may interfere with mating decisions (McGlothlin et al. 2004
It is not yet clear whether testosterone in females acts as an evolutionary constraint on the testosterone-mediated suite in males. The extent to which the sexes are genetically correlated in testosterone production is unknown. Our initial investigation of individual variation indicates that males and females may regulate testosterone in different ways. Whereas males respond to GnRH challenges throughout the breeding season, females seem to do so only when producing eggs (Jawor et al. 2007
). This effect has interesting implications for the role of female testosterone in maternal effects, as the magnitude of this GnRH challenge response showed a strong correlation with testosterone deposited in the yolk, but it suggests that the evolution of the testosterone-mediated suite may be somewhat decoupled across sexes. Further work, especially applying the approaches described in §4
to both sexes, is necessary to determine the importance of cross-sexual interactions in the evolution of hormone-mediated suites.