Love Is Blind: Indiscriminate Female Mating Responses to Male Courtship Pheromones in Newts (Salamandridae)

Internal fertilization without copulation or prolonged physical contact is a rare reproductive mode among vertebrates. In many newts (Salamandridae), the male deposits a spermatophore on the substrate in the water, which the female subsequently takes up with her cloaca. Because such an insemination requires intense coordination of both sexes, male newts have evolved a courtship display, essentially consisting of sending pheromones under water by tail-fanning towards their potential partner. Behavioral experiments until now mostly focused on an attractant function, i.e. showing that olfactory cues are able to bring both sexes together. However, since males start their display only after an initial contact phase, courtship pheromones are expected to have an alternative function. Here we developed a series of intraspecific and interspecific two-female experiments with alpine newt (Ichthyosaura alpestris) and palmate newt (Lissotriton helveticus) females, comparing behavior in male courtship water and control water. We show that male olfactory cues emitted during tail-fanning are pheromones that can induce all typical features of natural female mating behavior. Interestingly, females exposed to male pheromones of their own species show indiscriminate mating responses to conspecific and heterospecific females, indicating that visual cues are subordinate to olfactory cues during courtship.     

Sexual isolation between two newt species, Triturus vulgaris and T. montandoni (Amphibia, Urodela, Salamandridae)

In the present study we investigated sexual isolation between Triturus vulgaris and 77 montandoni in mating experiments run under semi-natural conditions. The two newt species offer a suitable model for studying evolution of reproductive isolation and mating preferences because they arc genetically the most similar species within the genus and readily hybridize in nature. Separate experiments were conducted in which groups of virgin females were placed together (in artificial pools) with groups of homospecific, heterospecific or both types of males. The estimates of reproductive isolation and mating propensity were based on the numbers of females producing hybrids and/or non-hybrid progeny. The levels of reproductive isolation, isolation asymmetry (IA) and propensity asymmetry (PA) were significant only for experiments in which females were given a choice between conspecific and heterospecific males. This implies that mating experiments with no interspecific choice may reduce discrimination and affect patterns of IA and PA. Asymmetry in reproductive isolation was also significant when the analysis was confined to just inseminated females. Differences in habitat preferences and condition of females possibly contributed to the relatively high values of PA.     

New(t)s and views from hybridizing MHC genes: introgression rather than trans-species polymorphism may shape allelic repertoires

One of the key features of major histocompatibility complex (MHC) genes is the frequent occurrence of trans-species polymorphism, that is 'the passage of allelic lineages from ancestral to descendant species' (Klein et al. 2007). Selectively maintained ancestral polymorphism may, however, be hard to distinguish from introgression of MHC alleles between hybridizing species (Fig. 1). In this issue of Molecular Ecology, Nadachowska-Brzyska et al. (2012) present data that suggest that the latter can be observed in two closely related species of newts, Lissotriton vulgaris (Lv) and L. montandoni (Lm) from south-east Europe. Strikingly, allelic MHC variation displayed more structure between geographically separated populations of L. vulgaris than across species in the hybrid zone. This suggests that high MHC variation in L. montadoni may result from mainly unidirectional gene flow between species, while differentiation between northern and southern populations of L. vulgaris might reflect local adaptation.     

Pheromones and body coloration affect mate recognition in the Japanese nine-spotted moth Amata fortunei (Lepidoptera: Arctiidae)

Adults of the Japanese nine-spotted moth, Amata fortunei, are diurnal and have white-spotted black wings and a black-and-yellow striped body pattern. We evaluated whether this species uses sex pheromones and whether visual cues from the female body are used in mate recognition. We introduced extracts of potentially scent-bearing abdominal tips of females to males. Males responded more to the extracts than to dried female specimens, suggesting the presence of sex pheromones in the extracts. Indeed, no males responded to the dried female specimens. To evaluate the importance of visual cues, we conducted the experiments with crude extract and an additional model stimulant. Males responded more to model females with the same number and similar area of yellow bands to the original conspecific females than to those with more bands and greater total band area, suggesting that dissimilarity in band number and area to conspecific females could interrupt male mating behavior. Males of A. fortunei likely find mating partners using olfactory cues over relatively long distances, while using both olfactory and visual cues over short distances. These results suggest that olfactory cues play a major role in mate recognition, whereas visual cues play a supplementary role.     

Conspecific and heterospecific pheromone effects on female receptivity

Courtship pheromones play an important role in salamander reproductive behaviour. In salamanders of the family Plethodontidae, males deliver specialized pheromones to females during courtship interactions. These courtship pheromones increase female receptivity and may be involved in mate discrimination. In order to test hypotheses related to mate discrimination, we staged courtship encounters between male-female Plethodon shermani pairs in which the female received pheromones obtained from either conspecific (P. shermani) or heterospecific (P. yonahlossee orP. montanus ) males. Both conspecific and heterospecific pheromones increased female receptivity. Moreover, pheromones from both heterospecific species were as effective as the conspecific pheromone in increasing female receptivity inP. shermani females. Our results suggest that the courtship pheromone signal and function may be conserved across related species, with mate discrimination occurring before pheromone delivery. Copyright 2003 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.     

Body Size and the Maintenance of Reproductive Isolation in Stickleback,Genus Gasterosteus

The blackspotted stickleback Gasterosteus wheatlandi and the widely studied threespine stickleback G. aculeatus are sympatric throughout the former’s range and share many aspects of life history and reproductive behaviour. These two species differ significantly in size, with G. wheatlandi of both sexes measured at approximately 60% of the standard length of their G. aculeatus counterparts. This study concentrated on G. wheatlandi courtship behaviour and investigated its role in the maintenance of reproductive isolation with G. aculeatus. Specifically, the roles that (1) female body size plays in influencing male courtship preferences and (2) male body size and behaviour play in female courtship preferences were investigated through dummy and live conspecific and heterospecific stimulus presentations. Male G. wheatlandi courtship preferences are consistent with previously described patterns for G. aculeatus. Males of both species preferentially approach and court the larger of two simultaneously presented live or dummy females. Thus, the smaller G. wheatlandi males are indiscriminate with respect to assortative mate choice; not only preferring to approach and court more fecund conspecific females but, more significantly, G. aculeatus‐sized females. In contrast, females of both species demonstrate strong assortative courtship preferences. When presented with pairs of flask‐enclosed males, females of both species preferentially orient and court the conspecific male over the heterospecific. Similarly, when presented with a conspecific male and a heterospecific male presented singly, females prefer to enter the nest of the conspecific. Systematic analysis of the interactions between these pairs of fish (one male, one female) demonstrates that the breakdown of courtship in heterospecific courtship occurs late in the courtship sequence when the widely differing forms of male leading behaviour results in drastically differing female responses. I suggest that, as previously described in G. aculeatus, the supernormality effect plays a significant role in mediating adaptive mate choice behaviour in G. wheatlandi. However, the added element of a larger sympatric species introduces a possible cost in time and energy devoted to courting heterospecific, and sympatric, females that the larger G. aculeatus do not likely incur. There is substantial evidence from many sympatric G. aculeatus species pairs that there is assortative mate choice based on size and/or courtship behaviour. Courtship trials suggest a more pervasive role for females in assortative mate choice. Whether it is male body size per se, or in combination with behaviour, morphology or other cues, is unresolved in the present study.     

The role of heliothine hairpencil compounds in female Heliothis virescens (Lepidoptera: Noctuidae) behavior and mate acceptance

Studies on numerous insect species suggest that male-produced sex pheromones play a role in attracting females; as aphrodisiacs, making females more quiescent; or as a means of inhibiting competing males. Male heliothine moths display abdominal hairpencils during courtship, but the specific effects of the odors released on female behavior have not yet been elucidated. This study investigates the role of male hairpencil compounds in female Heliothis virescens mating behavior. Female H. virescens were exposed to filter paper loaded with hairpencil extracts of male H. virescens, Heliothis subflexa and Helicoverpa zea, and observed for behavioral responses to odors. Single synthetic compounds found in the H. virescens hairpencil blend were also tested. In mating assays between single male and female H. virescens it was found that: (i) antennectomized females mated less frequently than sham-operated females; (ii) females mated less frequently with males whose hairpencils had been surgically removed; (iii) females mated with males with ablated hairpencils if a filter paper loaded with one male equivalent of H. virescens hairpencil extract was presented simultaneously; and (iv) this effect was species-specific, as presentation of H. subflexa or H. zea hairpencil extracts did not restore mate acceptance. This study suggests that odors released by male hairpencils are important in mate acceptance by female H. virescens, and may play a role in mate choice and species isolation.     

Phylogeography of two European newt species--discordance between mtDNA and morphology

The newts Triturus vulgaris and Triturus montandoni are sister species that exhibit contrasting levels of intraspecific morphological variation. Triturus vulgaris has a broad Eurasiatic distribution encompassing both formerly glaciated and unglaciated areas and shows substantial morphological differentiation in the southern part of its range, while T. montandoni, confined to the Carpathians, is morphologically uniform. We analysed sequence variation of two mtDNA fragments of the total length of c. 1850 bp in 285 individuals of both species collected from 103 localities. Phylogenetic analysis of 200 unique haplotypes defined 12 major clades, their age estimated at c. 4.5-1.0 million years (Myr). Most of the older clades were found in the southern part of the range, and also in central Europe, mainly in Romania. The distribution of mtDNA clades points to the existence of several glacial refugia, located in the Caucasus region, Anatolia, the Balkan Peninsula, Italy, and more to the north in central Europe. The concordance between mtDNA based phylogeny and the distribution of T. vulgaris subspecies was weak. Triturus montandoni haplotypes did not form a monophyletic group. Instead they were found in six clades, in five of them mixed with T. vulgaris haplotypes, most likely as a result of past or ongoing hybridization and multiple introgression of mtDNA from T. vulgaris to T. montandoni. Patterns of sequence variation within clades suggested long-term demographic stability in the southern groups, moderate and relatively old demographic growth in the populations inhabiting central Europe, and high growth in some of the groups that colonized northern parts of Europe after the last glacial maximum.     

Nuclear markers,mitochondrial DNA and male secondary sexual traits variation in a newt hybrid zone (Triturus vulgaris x T. montandoni)

The smooth and the Montandon's newts (Triturus vulgaris and T. montandoni) are genetically similar sister species with highly divergent male secondary sexual traits involved in complex courtship behaviour. Their parapatric ranges overlap at moderate elevations in the Carpathian Mountains where they hybridize readily. Here we present a detailed study of genetic and morphological variation in populations from the area of sympatry. Analysis of variation at seven nuclear markers, mtDNA and male sexual secondary traits was complemented with an ecological survey of breeding sites characteristics. Extensive hybridization was revealed with back-cross individuals similar to either parental species predominating among hybrids. The hybrid zone exhibited a mosaic pattern: the genetic composition of the populations was correlated only weakly with their geographical position. No association with habitat type was found. Departures from Hardy-Weinberg proportions, significant linkage disequilibria and bimodal distribution of genotypes suggest strongly that assortative mating is an important factor shaping the genetic composition of hybrid populations. The pattern of cytonuclear disequilibria did not indicate much asymmetry in interspecific matings. Changes in the frequency of nuclear markers were highly concordant, whereas mtDNA showed much wider bidirectional introgression with 14% excess of T. montandoni haplotype. We argue that the mosaic structure of the newt hybrid zone results mainly from stochastic processes related to extinction and recolonization. Microgeographical differences in mtDNA introgression are explained by historical range shifts. Since morphologically intermediate males were underrepresented when compared to hybrid males identified by genetic markers, sexual selection acting against the morphological intermediates is implied. We discuss the implications of these findings in the context of reinforcement of prezygotic isolation in newts.     

Sexual size and shape evolution in European newts (Amphibia: Caudata: Salamandridae) on the Balkan Peninsula

We used a phylogenetic perspective in an examination of the direction and extent of sexual dimorphism in body size and body shape in European newts from the Balkan Peninsula (alpine newts, Mesotriton alpestris; crested newts, Triturus cristatus superspecies; smooth newts, Lissotriton vulgaris). We found a strong, female‐biased sexual size dimorphism (SSD) in the analysed clades of alpine newt, whereas within crested newts we found a less stringent female‐biased SSD in Triturus carnifex, Triturus macedonicus and Triturus karelinii, and no significant SSD in T. cristatus or Triturus dobrogicus. Among the smooth newts, we found male‐biased SSD in Lissotriton vulgaris vularis and Lissotriton vulgaris greacus and no SSD in Lissotriton vulgaris meridionalis. Most of these newts also exhibit a significant sexual dimorphism in body shape, which varied more randomly than body size, regardless of SSD level. Female and male body size as well as the degree of SSD displayed statistically significant phylogenetic signal, while sexual dimorphism in body shape was phylogenetically independent. The relationship between independent contrast data for female size and male size indicated that SSD in European newts could be driven by a disproportionate increase in female size as increase in female size was not accompanied by a proportional increase in male size.     

Sequential mate choice by multiply mating smooth newts: females become more choosy

In some species, females sequentially mate with different maleswithin a single mating period, store sperm until the eggs areeventually fertilized, and gain no other resources from themales. Halliday hypothesized that, for such species, a femalecould ensure fertilization of her eggs by mating with the firstmale that she encounters; thereafter, she can maximize the qualityof her progeny by sampling further males and by mating onlywith males of higher quality than previously mated males. Thishypothesis predicts that females' choosiness will increase asthe breeding season progresses. We tested this prediction ofHalliday's hypothesis by examining mate choice by female smoothnewts (Triturus vulgaris vulgaris). Males of this species developa dorsal crest during the courtship season. Crest height variesbetween males and is potentially an indicator of male quality.Initially, female smooth newts were equally willing to matewith males with low or high crests. However, in their secondmating, females remated only with high-crested males. Theseresults support our prediction and are consistent with Halliday'shypothesis. If crest height in smooth newts is an indicatorof quality, this change in females' mate choice criterion allowsfemales both to initiate egg-laying very early in the seasonand, subsequently, to mate preferentially with higher qualitymates     

Genetic and behavioral components of the cryptic species boundary between Laupala cerasina and L. kohalensis (Orthoptera: Gryllidae)

Cryptic species are often hypothesized on the basis of differences in courtship signals. These signal differences suggest that mate recognition systems, which include both courtship signals and responses to those signals, have diverged between genetically isolated populations. Cryptic species are therefore thought to represent distinct genetic units, the boundaries of which are maintained by premating incompatibilities, specifically by receiver preferences for conspecific signals. Laupala cerasina and L. kohalensis are sympatric species of swordtail crickets endemic to the big island of Hawaii, that are distinguishable by differences in male courtship song. We first tested whether groupings hypothesized by acoustic similarity reflect genetic groupings, using AFLP data to estimate genetic relationships. Second, we tested whether genetic boundaries are maintained by female preferences for conspecific song characteristics. Phonotaxis trials were used to determine the extent of female preferences for conspecific male song. Results generally support both hypotheses, but suggest the presence of porous species boundaries.     

Behavioural evidence for a female sex pheromone in Cotesia flavipes (Hymenoptera: Braconidae)

Abstract.  The mating behaviour of Cotesia flavipes Cameron, 1891 (Hymenoptera: Braconidae) was observed under laboratory conditions. The results demonstrate that chemical stimuli play an important role in bringing males and females together for mating. Females from this species release chemical compounds to attract males, which exhibit a distinct courtship behaviour divided into three main steps: (i) antennation, (ii) lateral bouncing and (iii) wing fluttering. Ethological tests using hexane extracts from two different parts of the female's body (head plus thorax and abdomen) demonstrate that males are more attracted and also display a courtship behaviour when a filter paper containing hexane extracts from the abdomen is presented to them, indicating that the abdomen is the source of the sex pheromone of this species. Naive males of this species respond to hexane extracts of this gland by displaying courtship behaviour. Furthermore, additional tests show that C. flavipes females mate only once, whereas virgin males do not show any preference for virgin or mated females, suggesting that mated females of this species continue to produce sex attractants after mating.     

Genetic dissimilarity predicts paternity in the smooth newt (Lissotriton vulgaris)

Under sperm competition, paternity is apportioned by polyandrous females according to the order of matings and the genetic quality of the inseminating males. In order to distinguish between these two effects, we sequentially paired 12 female smooth newts (Lissotriton vulgaris) with each of two males and, where possible, repeated the same procedure in reverse order of the identical males after assumed sperm depletion. For a total of 578 offspring, amplified fragment length polymorphisms genetic markers revealed multiple paternities in all matings, without significant first- or second-male sperm precedence. The paternity share of individual males was transitive across the two trials with male order switch, and successful males had a significantly higher genetic dissimilarity to the female than expected by chance. We argue that patterns of paternity in natural newt populations are determined through a combination of good genes and relatedness.     

Cross-fostering Does Not Influence the Mate Preferences and Territorial Behaviour of Males in Lake Victoria Cichlids

The ability to recognise conspecifics in contexts of mate choice and territorial defence may have large effects on an individual's fitness. Understanding the development of assortative behaviour may shed light on how species assortative behaviour evolves and how it may influence reproductive isolation. This is the case not only for female mate preferences, but also for male mate preferences and male territorial behaviour. Here we test with a cross-fostering experiment whether early learning influences male mate preferences and male–male aggression biases in two closely related, sympatrically occurring cichlid species Pundamilia pundamilia and Pundamilia nyererei from Lake Victoria. Males that had been fostered, either by a conspecific female or by a heterospecific female, were tested for their aggression bias, as well as for their mate preferences, in two-way choice tests. Males cross-fostered with conspecific and heterospecific foster mothers selectively directed their aggression towards conspecific intruders. The cross-fostering treatment also did not affect male mate preferences. These results are in striking contrast with the finding that females of these species show a sexual preference for males of the foster species.     

Mate Choice by Both Sexes Maintains Reproductive Isolation in a Species Flock of Pupfish (Cyprinodon spp) in the Bahamas

Female and male mate choices can reinforce reproductive isolation after sympatric speciation. Using a binary choice design, we examine the importance of visual cues in female mate choice in all three sympatric species of pupfish on San Salvador Island. We also examine the importance of olfactory cues in female choice of the hard‐shelled invertebrate specialist (Cyprinodon brontotheroides). We examine male mate choice in two of the three species, the scale eater (C. desquamator) and the detritivore (C. variegatus). Females of all three species use visual cues and prefer conspecific males. C. brontotheroides females do not use olfactory cues to discriminate between conspecific and heterospecific males. Males of C. desquamator and C. variegatus also preferentially court conspecific females. Thus, mutual mate choice, where both females and males exhibit mate choice, acts as a strong behavioral pre‐mating isolation mechanism in these sympatrically speciated pupfish.     

Reproductive isolation inProkelisia planthoppers (Homoptera: Delphacidae): Acoustic differentiation and hybridization failure

Males and females of Prokelisia marginata (Van Duzee) and Prokelisia dolusWilson communicate through substrate-transmitted vibrations. The acoustic signals (attraction and courtship calls) of these planthoppers are effective in mate location, attraction, and mate choice. Attraction calls are structurally distinct for both species and differ in pulse type, pulse repetition rate, and pulse duration. Using playback of prerecorded calls, individuals discriminated between conspecific and heterospecific signals. Depending on the sex and species, response calls were produced three to eight times more frequently to conspecifics than to heterospecifics. However, acoustic signals alone did not explain reproductive isolation and hybridization failure in these two congeners. Some heterospecific pairs called, courted, and attempted to join genitalia, but no connections were successful and no progeny were produced. Thus, acoustic behavior is not a guaranteed premating isolating mechanism in no-choice situations. Other courtship behaviors and possibly morphological differences in genitalia also contributed to their isolation. Females displayed a variety of rejection behaviors to conspecific and heterospecific males, suggesting that sexual selection (female choice), in addition to species recognition, may be an important force in the evolution of the acoustic signals of planthoppers. Although signal structure was not dependent on wing form (planthoppers exhibit wing dimorphism), the age when females first began to call was related to wing form. Brachypterous (flightless) females of both species began calling early in adult life (day 2), whereas macropterous (migratory) females began calling later in adult life (day 6). This pattern is consistent with the oogenesis-flight syndrome, in which reproductive maturity is delayed until after migration occurs.     

The role of silk in courtship and chemical communication of the false widow spider, <Emphasis Type="Italic">Steatoda grossa</Emphasis> (Araneae: Theridiidae)

In spiders, sex pheromones are often associated with silk produced by females, and function in mate attraction, recognition, and evaluation. Silk-bound pheromones typically elicit courtship behaviour in web-building spiders. Here we (1) describe courtship interactions of Steatoda grossa males with virgin or mated females, and (2) show that silk and methanol extracts of silk produced by virgin females trigger courtship behaviour (silk production) by males, whereas silk of mated females does not. Our results indicate that (1) virgin females produce a silk-bound sex pheromone, (2) males discriminate between virgin and mated females based on silk cues, and (3) male silk likely functions in sexual communication.