Loss of Avian Intromittent Organs as a Sperm Competition Strategy: A Race to Be Last.

AbstractThe loss of the intromittent organ (IO) in the majority of birds remains unexplained. Here, I propose that IO loss results from sperm competition in the context of the unique avian sperm storage system. The first stage of fertilization is the movement of sperm through the vagina from the site of ejaculation to the sperm storage tubules (SSTs) at the uterovaginal junction. In a second stage, sperm are released from the SSTs and move through the uterus to the site of fertilization in the infundibulum. Last-male fertilization precedence can occur if sperm that arrive later at the uterovaginal junction occupy uterus-side SSTs, which then have a head start in the race to the infundibulum with each ovulation. Under such "last-in, first-out" conditions, there is strong sperm competition to arrive later at the SSTs. Consequently, the optimal male strategy would be placement of the ejaculate at the cloacal opening to allow any other sperm in the vagina to reach the SSTs first. Cloacal placement is effectively achieved by loss of the IO. The evolution of altricial development in Neoaves, the largest clade that lacks IOs, created conditions that favor IO loss. Specifically, the smaller clutch sizes and hatching asynchrony of altricial birds increase the intensity of sperm competition for fertilization of early eggs in the laying sequence and thus the selective advantage of later arrival at the SSTs. The rarity of IO loss among all animals suggests that the complex mechanism of avian fertilization creates unique conditions for sperm competition.

Divergence of a speciation trait through artificial selection: Insights into constraints, by-product effects and sexual isolation.

Speciation and sexual isolation often occur when divergent female mating preferences target male secondary sexual traits. Despite the importance of such male signals, little is known about their evolvability and genetic linkage to other traits during speciation. To answer these questions, we imposed divergent artificial selection for 10 non-overlapping generations on the Inter-Pulse-Interval (IPI) of male courtship songs; which has been previously shown to be a major species recognition trait for females in the Drosophila athabasca species complex. Focusing on one of the species, Drosophila mahican (previously known as EA race), we examined IPI's: (1) rate of divergence, (2) response to selection in different directions, (3) genetic architecture of divergence and (4) by-product effects on other traits that have diverged in the species complex. We found rapid and consistent response for higher IPI but less response to lower IPI; implying asymmetrical constraints. Genetic divergence in IPI differed from natural species in X versus autosome contribution and in dominance, suggesting that evolution may take different paths. Finally, selection on IPI did not alter other components of male songs, or other ecological traits, and did not cause divergence in female preferences, as evidenced by lack of sexual isolation. This suggests that divergence of male courtship song IPI is unconstrained by genetic linkage with other traits in this system. This lack of linkage between male signals and other traits implies that female preferences or ecological selection can co-opt and mould specific male signals for species recognition free of genetic constraints from other traits.

The effect of short-term exposure to high temperatures on male courtship behaviour and mating success in the fruit fly Drosophila virilis.

Human-induced climate change is leading to higher average global temperatures and increasingly extreme weather events. High temperatures can have obvious effects on animal survival, particularly in ectotherms. However, the temperature at which organisms become sterile may be significantly lower than the temperature at which other biological functions are impaired. In the fruit fly Drosophila virilis, males are sterilized at temperatures above 34 °C, but are still active and able to mate normally. We investigated the male behavioural changes associated with high-temperature fertility loss. We exposed males to a warming treatment of 34.4 °C or 36.6 °C for 4 h, and then recorded their mating behaviour after being allowed to recover for 24 h. Previous work in this species suggests that males exposed to 34.4 °C lose the ability to produce new sperm, but can utilize mature sperm produced before the heat shock. We therefore predicted that these males would increases their courtship rate, and reduce their choosiness, in order to try to ensure a mating before their remaining mature sperm die. In contrast, over two-thirds of males exposed to 36.6 °C are completely sterile. In standard mating trials, earlier exposure to 34.4 °C or 36.6 °C did not affect male courtship behaviour when compared to control males kept at 23 °C. Exposure to high temperatures also did not alter the extent to which males directed courtship toward females of the same species. However, males exposed to 36.6 °C were significantly slower to mate, and had a reduced likelihood of mating, when compared to control males. Overall, exposure to high temperatures did not alter male courtship behaviour, but did lower their likelihood of mating. This suggests that females can distinguish between normal and heat-sterilized males before mating, and that female mate choice may at least partly mitigate the population-level consequences of high-temperature induced male sterility in this species.

Individuality, species-specific features, and female discrimination of male southern white rhinoceros courtship calls.

Male vocalizations associated with courtship can play a key role in mate selection. They may help females obtain information about males' quality and identity and/or may contain species-specific properties that help prevent interspecies breeding. Despite vocalizations being a prominent part of the courtship of white rhinos, the role that they play in white rhino breeding behaviour has not been extensively studied. Both southern (SWR) and critically endangered northern white rhino (NWR) males intensively vocalize during courtship with hic calls. We examined these calls and found that call properties differed between NWR and SWR males. In addition, we found that individual SWR males could be identified with a high degree of accuracy using their hic calls and that the signature information capacity in hic calls would allow females to individually recognize about 11 adult males living in or moving through their home-ranges, which may help with mate selection. Then, we conducted playback experiments with wild anoestrus SWR females. The females discriminated between the NWR and SWR hic calls and between the SWR hic and SWR pant calls. However, we only found differences in the latency of observed behaviours, not in their duration or in the intensity of females' reaction. This might suggest that females which are not in oestrus are not highly responsive to a male's motivation (i.e., seeking contact or mating), but are more interested in assessing his dominance status or familiarity. Ultimately, our results indicate that courtship hic calls encode information which might help females choose mating partners.

Commentary on Kehl et al. "Young male mating success is associated with sperm number but not with male sex pheromone titres": Unnatural experimental conditions inflate the importance of male courtship activity on mating success in a butterfly.

Over the last years, several studies suggested that male courtship activity is more important than female preference for male secondary sexual traits in determining male mating success in the butterfly Bicyclus anynana. We use Kehl et al. (Front Zool 12, 2015)'s study and related publications, to highlight three methodological and conceptual aspects of laboratory experiments that distort the social environment compared to natural conditions. We argue that such experimental biases prevent the expression of female mate choice and artificially inflate the role of male activity in determining mating success. We really want to stress that any work performed in laboratory conditions using extreme cage densities or sizes impedes female mate choice and promotes male-male competition when sexual conflict occurs about mating decisions. Hence, such studies, and the derived conclusions, are only applicable to ecologically-irrelevant conditions and cannot be extrapolated to more natural laboratory or field conditions. Our concerns may be relevant to many behavioural studies quantifying sexual selection across taxa. This commentary adds to the increasing scientific awareness that: i) mating outcome is, across taxa, the result of a sexual conflict whose outcome is under female, and not male, control; ii) the social environment used to quantify mating success is of utmost importance to produce reliable estimates of the strength and the direction of sexual selection on sexually-selected traits, as they evolve in nature.

A reply to Nieberding and Holveck: beyond experimental design and proximate mechanisms - mate choice in the face of sexual conflict.

We summarise our work on male mating behaviour in the tropical butterfly Bicyclus anynana, responding to the commentary provided by Nieberding and Holveck. We acknowledge that our laboratory studies are not free of shortcomings and potential caveats, though we attempted to address or highlight these within each paper. The concerns raised seem to stem mainly from different notions with respect to the proximate basis of old male mating advantage, and specifically the relative importance of male behaviour versus pheromone blend. In our view, our experiments provided compelling evidence for a prominent role of male behaviour, while we were unable to obtain clear evidence for a major role of male sexual pheromones. In addition to the lack of evidence we argue that a preference of females for older males based on pheromone blend is unlikely, as pheromone titres do not seem to indicate male quality and, more importantly, females actually suffer a fitness cost when mating with older males. The latter suggests that old male mating advantage arises from sexual conflict rather than cooperation. We thus highlight the importance of considering both the proximate and the ultimate level for gaining an integrative understanding of complex behavioural patterns.

Deposition, removal and production site of the amorphous mating plug in the spider Philodromus cespitum.

In order to avoid sperm competition, males of many taxa apply physical barriers, so-called mating plugs, into female genitalia. Females may control which males deposit a plug through pre-copulatory mate choice or by influencing plug efficacy to avoid costs imposed by plugging. However, subsequent suitors might remove the plugs. We investigated behavioural and morphological aspects of plug deposition and removal in a promiscuous spider, Philodromus cespitum (Philodromidae). We performed mating trials to investigate factors affecting plugging. To identify the plug origin, we conducted a morphological analysis using 3D X-ray microtomography and histology of the male copulatory organ and the female genital tract. In P. cespitum, the plug material is produced in the male genital bulb and transferred to the female together with sperm. The copulation is brief and terminated by the female. After mating, plugging material was found in the genital atrium of all females, covering it to a varying degree (10-100%). The extent of coverage was associated with the duration of movements of male copulatory organ connected with sperm transfer (i.e. full haematodochal expansions) and with the number of taps a male delivered with his legs to the female during courtship. Males larger than the female performed more tapping movements. Mating trials with plugged females revealed that males could remove plugs partly or entirely. Removal success increased with increasing foreleg length ratio between the male who removed the plug and the one who deposited it. We discuss our results in the light of the potential female control of plug deposition and removal.

Female Mate Choice Based on Male Nuptial Coloration in Pale Chub, Zacco platypus.

Males of many animals exhibit conspicuous secondary sexual traits, which are often the result of sexual selection. During the breeding period of the pale chub (Pisces: Cyprinidae), males show various sexual traits, such as long anal fins, breeding tubercles on the head, and/or conspicuous red and greenish-blue nuptial colorations. Although these conspicuous sexual dimorphisms and dichromatisms indicate that this can serve as a model species for understanding sexual selection, it is unclear whether these male sexual traits are related to female mate choice. This study examined the relationships between female mate choice and male sexual traits under laboratory conditions. In a dichotomous choice experiment, no relationship was found between female preference and male body size, anal fin length or number of breeding tubercles. In addition, female preference was not detected for traits related to the greenish-blue nuptial color of males. Instead, females chose males based on the degree of expression of red nuptial coloration. Such female preference may be useful for selecting high-quality males with good foraging abilities or good immune function related to carotenoids and sexual hormones. The present study suggests that red nuptial coloration of the male pale chub evolved in part through female mate choice.

Egg viability decreases rapidly with time since ovulation in the rainbow darter Etheostoma caeruleum: implications for the costs of choosiness.

Egg viability in the rainbow darter Etheostoma caeruleum, a fish apparently lacking female mate choice, was found to decline rapidly after ovulation. It was observed that the majority of a female's clutch may fail to hatch if she is prevented from mating for as little as 6 h. These data suggest that exercising female mate preferences may be selectively disfavoured in E. caeruleum due to the high cost of delaying mating.

Can MHC-assortative partner choice promote offspring diversity? A new combination of MHC-dependent behaviours among sexes in a highly successful invasive mammal.

Sexual selection involving genetically disassortative mate choice is one of several evolutionary processes that can maintain or enhance population genetic variability. Examples of reproductive systems in which choosers (generally females) select mates depending on their major histocompatibility complex (MHC) genes have been reported for several vertebrate species. Notably, the role of MHC-dependent choice not in mating contexts, but in other kinds of social interactions such as in the establishment of complex social systems, has not yet drawn significant scientific interest and is virtually absent from the literature. We have investigated male and female MHC-dependent choice in an invasive population of North American raccoons (Procyon lotor) in Germany. Both male and female raccoons rely on olfaction for individual recognition. Males have an unusually complex social system in which older individuals choose unrelated younger ones to form stable male coalitions that defend territories and a monopoly over females. We have confirmed that females perform MHC-disassortative mate choice and that this behaviour fosters genetic diversity of offspring. We have also observed that males build coalitions by choosing male partners depending on their MHC, but in an assortative manner. This is the first observation of antagonistic MHC-dependent behaviours among sexes. We show that this is the only combination of MHC-dependent partner choice that leads to outbreeding. In the case of introduced raccoons, such behaviours can act together to promote the invasive potential of the species by increasing its adaptive genetic divergence.

Genetic population structure in an equatorial sparrow: roles for culture and geography.

Female preference for local cultural traits has been proposed as a barrier to breeding among animal populations. As such, several studies have found correlations between male bird song dialects and population genetics over relatively large distances. To investigate whether female choice for local dialects could act as a barrier to breeding between nearby and contiguous populations, we tested whether variation in male song dialects explains genetic structure among eight populations of rufous-collared sparrows (Zonotrichia capensis) in Ecuador. Our study sites lay along a transect, and adjacent study sites were separated by approximately 25 km, an order of magnitude less than previously examined for this and most other species. This transect crossed an Andean ridge and through the Quijos River Valley, both of which may be barriers to gene flow. Using a variance partitioning approach, we show that song dialect is important in explaining population genetics, independent of the geographic variables: distance, the river valley and the Andean Ridge. This result is consistent with the hypothesis that song acts as a barrier to breeding among populations in close proximity. In addition, songs of contiguous populations differed by the same degree or more than between two populations previously shown to exhibit female preference for local dialect, suggesting that birds from these populations would also breed preferentially with locals. As expected, all geographic variables (distance, the river valley and the Andean Ridge) also predicted population genetic structure. Our results have important implications for the understanding whether, and at what spatial scale, culture can affect population divergence.

Females can solve the problem of low signal reliability by assessing multiple male traits.

Male signals that provide information to females about mating benefits are often of low reliability. It is thus not clear why females often express strong signal preferences. We tested the hypothesis that females can distinguish between males with preferred signals that provide lower and higher quality direct benefits. In the field cricket, Gryllus lineaticeps, females usually prefer higher male chirp rates, but chirp rate is positively correlated with the fecundity benefits females will receive from males only for males that have experienced low quality diets. We paired females with muted males that were maintained on low or high nutrition diets, during the interactions we broadcast a replacement high chirp rate, and we observed whether females mated with the assigned male. Females were more likely to mate when paired with low nutrition males. These results suggest that females have evolved assessment mechanisms that allow them distinguish between males with preferred signals that provide high quality benefits (low nutrition males with high chirp rates) and males with preferred signals that provide low quality benefits (high nutrition males with high chirp rates).

Sexual Selection on male cuticular hydrocarbons via male-male competition and female choice.

Traditional views of sexual selection assumed that male-male competition and female mate choice work in harmony, selecting upon the same traits in the same direction. However, we now know that this is not always the case and that these two mechanisms often impose conflicting selection on male sexual traits. Cuticular hydrocarbons (CHCs) have been shown to be linked to both social dominance and male attractiveness in several insect species. However, although several studies have estimated the strength and form of sexual selection imposed on male CHCs by female mate choice, none have established whether these chemical traits are also subject to sexual selection via male-male competition. Using a multivariate selection analysis, we estimate and compare sexual selection exerted by male-male competition and female mate choice on male CHC composition in the broad-horned flour beetle Gnatocerus cornutus. We show that male-male competition exerts strong linear selection on both overall CHC abundance and body size in males, while female mate choice exerts a mixture of linear and nonlinear selection, targeting not just the overall amount of CHCs expressed but the relative abundance of specific hydrocarbons as well. We discuss the potential implications of this antagonistic selection with regard to male reproductive success.

Evolution of the ungulate dewlap: thermoregulation rather than sexual selection or predator deterrence?

BACKGROUND: Dewlaps are iconic features of several ungulate species and, although a role in signalling has been postulated, their function remains largely unexplored. We recently failed to find any age-independent link between dewlap size and social status in the common eland (Tragelaphus oryx), pointing to the possibility that sexual selection may not be the primary cause of dewlap evolution in ungulates. Here I use a two-pronged approach to test hypotheses on the function of ungulate dewlaps: an interspecific comparative analysis of bovids and deer, and an intraspecific study of eland antelopes in the wild. RESULTS: Across species, the presence of dewlaps in males was not found to be associated with sexual size dimorphism, a commonly used measure of the intensity of sexual selection. The presence of dewlaps was, however, linked to very large male body size (>400 kg), which agrees with a thermoregulatory function as lower surface/volume-ratio counteracts heat dissipation in large-bodied species. In eland antelopes, large dewlap size was associated with higher, rather than lower, incidence of claw-marks (independently of age), a result which speaks against the dewlap as a predator deterrent and rather indicates a predation cost of the structure. CONCLUSION: The findings suggest that, although an additional function in communication should not be ruled out, the dewlap of ungulates may contrast with that of lizards and birds in thermoregulation being a primary function.

The vibrational signals that male fiddler crabs (Uca lactea) use to attract females into their burrows.

In some fiddler crab species, males emit vibrations from their burrows to mate-searching females after they have attracted a female to the burrow entrance using a waving display. Although the vibrations are considered acoustic signals to induce mating, it has not been demonstrated whether the vibrations attract the females into the burrow and, consequently, influence females' mating decisions. We investigated the structures and patterns of the vibrations using a dummy female and demonstrated experimentally a female preference for male vibrations in Uca lactea in the field. The acoustic signals consisted of repetitions of pulses. The dominant frequency of the pulses decreased with male carapace width. The pulse length decreased slightly with an increasing number of vibrational repetitions, and the pulse interval increased with increasing repetitions. These factors imply that the vibrations convey information on male characteristics, such as body size and stamina. In the experiment on female mate choice, the females significantly preferred males with higher pulse repetition rates when they were positioned at the entrance of the burrow, indicating that the females use the male vibrational signals to decide whether to enter the burrow. However, females showed no preference for the vibrations once they were inside a burrow, i.e., whether they decided to copulate, suggesting that the vibrations do not independently affect a female's final decision of mate choice. The vibrations inside the burrow might influence a female's decision by interaction with other male traits such as the burrow structure.

Mating patterns of female Leon Springs pupfish Cyprinodon bovinus.

In a field study of Leon Springs pupfish Cyprinodon bovinus, two questions about female promiscuity were investigated. First, were females selective in the males with whom they spawned or were they unselective, spawning randomly among males? Second, how promiscuous were the females, i.e. with how many males did they spawn? If simply spawning with many males maximized a female's reproductive success, then females might be expected to spawn randomly with as many males as possible. Alternatively, if females were selective but engaged in multiple mating, they would limit their spawning to preferred males. In the only wild population of this endangered fish, breeding males defend closely associated territories in the shallow margins of a single desert pool. No territories were observed elsewhere in the pool. Therefore, all territorial males were present simultaneously and females could survey all of them, depositing any number of eggs with one, a few or many males. Rather than spawning randomly, females surveyed many males first, visited relatively few males and ultimately spawned with a small fraction of those available males. With increasing numbers of spawns, however, females increased the number of different mates with whom they spawned. Thus, females showed a bet-hedging tactic of having a narrow mate preference while also laying eggs in the territories of other males, possibly to reduce egg predation and to avoid inbreeding.

Female mate choice predicts paternity success in the absence of additive genetic variance for other female paternity bias mechanisms in Drosophila serrata.

After choosing a first mate, polyandrous females have access to a range of opportunities to bias paternity, such as repeating matings with the preferred male, facilitating fertilization from the best sperm or differentially investing in offspring according to their sire. Female ability to bias paternity after a first mating has been demonstrated in a few species, but unambiguous evidence remains limited by the access to complex behaviours, sperm storage organs and fertilization processes within females. Even when found at the phenotypic level, the potential evolution of any mechanism allowing females to bias paternity other than mate choice remains little explored. Using a large population of pedigreed females, we developed a simple test to determine whether there is additive genetic variation in female ability to bias paternity after a first, chosen, mating. We applied this method in the highly polyandrous Drosophila serrata, giving females the opportunity to successively mate with two males ad libitum. We found that despite high levels of polyandry (females mated more than once per day), the first mate choice was a significant predictor of male total reproductive success. Importantly, there was no detectable genetic variance in female ability to bias paternity beyond mate choice. Therefore, whether or not females can bias paternity before or after copulation, their role on the evolution of sexual male traits is likely to be limited to their first mate choice in D. serrata.

Long-term video and genetic data yield insights into complex sociality of a solitary large carnivore.

American black bears (Ursus americanus) may be more social than currently understood. We used long-term video and genetic data to evaluate social interactions among wild, independent-aged black bear on a conservation property in western Montana, USA. We used multinomial logistic regression to evaluate predictions about male-male interactions within the context of individual fitness, female-female interactions within the context of inclusive fitness, and male-female interactions within the context of female counterstrategies to infanticide. Overall, our findings challenged the assumption that independent-aged bears interact only during the mating season or when concentrated feeding sites are present. We documented 169 interaction events by at least 66 bear pairs, 92 (54%) of which occurred outside of the peak mating season and in the absence of concentrated feeding sites. The probability that male-male pairs engaged in play and other non-agonistic behaviours was higher than that for female-female pairs. Conversely, the probability that female-female pairs engaged in chase behaviour was higher than that for male-male and male-female pairs. We documented evidence of female mate choice, female resource defense, sexually selected infanticide (SSI), and female counterstrategies to avoid SSI. Our findings improve our understanding of ursid ethology and underscore the complexity of ursid sociality.

Reproductive isolation and patterns of genetic differentiation in a cryptic butterfly species complex.

Molecular studies of natural populations are often designed to detect and categorize hidden layers of cryptic diversity, and an emerging pattern suggests that cryptic species are more common and more widely distributed than previously thought. However, these studies are often decoupled from ecological and behavioural studies of species divergence. Thus, the mechanisms by which the cryptic diversity is distributed and maintained across large spatial scales are often unknown. In 1988, it was discovered that the common Eurasian Wood White butterfly consisted of two species (Leptidea sinapis and Leptidea reali), and the pair became an emerging model for the study of speciation and chromosomal evolution. In 2011, the existence of a third cryptic species (Leptidea juvernica) was proposed. This unexpected discovery raises questions about the mechanisms preventing gene flow and about the potential existence of additional species hidden in the complex. Here, we compare patterns of genetic divergence across western Eurasia in an extensive data set of mitochondrial and nuclear DNA sequences with behavioural data on inter- and intraspecific reproductive isolation in courtship experiments. We show that three species exist in accordance with both the phylogenetic and biological species concepts and that additional hidden diversity is unlikely to occur in Europe. The Leptidea species are now the best studied cryptic complex of butterflies in Europe and a promising model system for understanding the formation of cryptic species and the roles of local processes, colonization patterns and heterospecific interactions for ecological and evolutionary divergence.

Pre-copulatory choices drive post-copulatory decisions: mechanisms of female control shift across different life stages.

BACKGROUND: The 'wallflower' hypothesis proposes females mate indiscriminately to avoid reproductive delays. Post-copulatory mechanisms may then allow 'trading up', favouring paternity of future mates. We tested links between pre- and post-copulatory choice in Latrodectus geometricus female spiders paired sequentially with two males. These females copulate as adults or as subadults and store sperm in paired spermathecae. Choosy adults have a higher risk of delays to reproduction than subadults. RESULTS: We predicted low pre-copulatory, but high post-copulatory choice at first matings for adults and the opposite for subadults. At second matings, we expected all females would prefer males superior to their first. We found all females mated indiscriminately at their first pairing, but in contrast to subadults, adults usually allowed only a single insertion (leaving one of their paired spermatheca empty); a mechanism of post-copulatory choosiness. Adult-mated females were more likely to remate than subadult-mated females when they became adults, showing a preference for larger males, while subadult-mated females tended to prefer males of greater size-corrected mass. CONCLUSIONS: Our results show that the 'wallflower' effect and 'trading up' tactics can be utilized at different life stages, allowing females to employ choice even if rejecting males is costly.