Assessment of Predation Risk by Juvenile Yellow Perch, Perca flavescens: Responses to Alarm Cues from Conspecifics and Prey Guild Members

In two laboratory experiments we tested juvenile yellow perch, Perca flavescens, for behavioural responses to alarm cues of injured conspecifics and several prey guild members: adult perch, Iowa darters, Etheostoma exile and spottail shiners, Notropis hudsonius. Spottail shiners are phylogenetically distant to yellow perch whereas Iowa darters and perch are both members of the Family Percidae. Groups of juvenile yellow perch increased shoal cohesion and movement towards the substrate after detecting conspecific alarm cues when compared to cues of injured swordtails, Xiphophorus helleri, a species phylogenetically distant from perch. Individual juvenile perch increased shelter use and froze more when exposed to chemical alarm cues from both juvenile and adult perch, shiners and darters compared to exposure to injured swordtail cues or distilled water. The response to cues of darters may indicate that alarm cues are evolutionarily conserved within percid fishes or that perch had learned to recognize darter cues. The response to spot tail shiners likely represents learned recognition of the cues of a prey guild member.     

Territorial behaviour of the male johnny darter,Etheostoma nigrum

Synopsis A field study was undertaken to describe the territorial behaviour of male johnny darters (Etheostoma nigrum) over the reproductive cycle. While defending eggs, males performed fewer Lateral Displays and lost fewer encounters with conspecific males than in the pre-egg phase. As the reproductive cycle progressed males were less responsive to females and performed relatively fewer courtship and more aggressive displays. Nesting males apparently shifted from a sexual to a parental phase shortly after acquiring eggs.Frequency of response, responsiveness (number of responses/number of intrusions x 100) and maximum distance of response (MDR) of male johnny darters were highest early in the reproductive cycle and decreased over the egg-guarding phase. The patterns of territorial defence exhibited by male johnny darters over the reproductive cycle were similar to those of other species of fish with paternal care.     

Male orangethroat darters,Etheostoma spectabile, do not prefer larger females

Synopsis Male orangethroat darters,Etheostoma spectabile, did not show a preference for larger females as measured by the number of matings or time until mating in a field enclosure experiment. The lack of male preference for female size may be due to the male-biased sex ratio at the site (seven males per female) or to low variation in the number of eggs deposited per spawning.     

Reproductive isolation between two darter species is enhanced and asymmetric in sympatry

Robust reproductive isolation was found between the rainbow darter Etheostoma caeruleum and the orangethroat darter Etheostoma spectabile, as more offspring were produced when conspecific males and females were crossed as compared with heterospecific crosses. Furthermore, fewer eggs resulted from heterospecific crosses involving sympatric E. spectabile females than those using allopatric E. spectabile females, while a similar pattern was not observed in heterospecific crosses using E. caeruleum females. These results suggest that reinforcement, i.e. selection for pre‐zygotic reproductive barriers driven by reduced hybrid fitness, may have contributed to the evolution and maintenance of reproductive barriers between these potentially hybridizing species in sympatry.     

Male and female contributions to behavioral isolation in darters as a function of genetic distance and color distance

Determining which reproductive isolating barriers arise first between geographically isolated lineages is critical to understanding allopatric speciation. We examined behavioral isolation among four recently diverged allopatric species in the orangethroat darter clade (Etheostoma: Ceasia). We also examined behavioral isolation between each Ceasia species and the sympatric rainbow darter Etheostoma caeruleum. We asked (1) is behavioral isolation present between allopatric Ceasia species, and how does this compare to behavioral isolation with E. caeruleum, (2) does male color distance and/or genetic distance predict behavioral isolation between species, and (3) what are the relative contributions of female choice, male choice, and male competition to behavioral isolation? We found that behavioral isolation, genetic differentiation, and male color pattern differentiation were present between allopatric Ceasia species. Males, but not females, discerned between conspecific and heterospecific mates. Males also directed more aggression toward conspecific rival males. The high levels of behavioral isolation among Ceasia species showed no obvious pattern with genetic distance or male color distance. However, when the E. caeruleum was included in the analysis, an association between male aggression and male color distance was apparent. We discuss the possibility that reinforcement between Ceasia and E. caeruleum is driving behavioral isolation among allopatric Ceasia species.     

Behavioral Isolation Based on Visual Signals in a Sympatric Pair of Darter Species

Elaborate visual communication signals characterize many animal lineages. Often sex‐limited, these signals are generally assumed to result from sexual selection, and in many cases, their evolution is thought to play a central role in speciation. The co‐evolution of male visual signals and female preferences is hypothesized to result in behavioral isolation between divergent lineages; however, for many lineages characterized by elaborate visual signals, the importance of visual differences in behavioral isolation is not well established. Darters (fish genus Etheostoma) are particularly appropriate for examining the role of visual signals in behavioral isolation. They comprise one of the most diverse groups of North American freshwater fish, and nearly every species is characterized by unique nuptial coloration. Multiple darter species co‐exist in sympatric populations, indicating that reproductive barriers are central to maintaining these extraordinarily diverse color patterns. This study demonstrates the presence of behavioral isolation between a pair of distinctly colored sympatric darter species, Etheostoma barrenense and Etheostoma zonale, through experimental observations using an artificial stream. In addition, a series of dichotomous mate‐choice trials indicate that females prefer conspecific males over heterospecifics based on visual differences alone. We therefore provide the first evidence that visual signals are a critical trait maintaining behavioral isolation in darters, a lineage of fishes with spectacular diversification in visual communication.     

Friends in Low Places: Responses of a Benthic Stream Fish to Intra‐Prey‐Guild Alarm Cues

Many aquatic species produce chemical alarm cues that serve as a warning to nearby conspecifics. In mixed‐species aggregations, individuals may also benefit by ‘eavesdropping’ on the chemical alarm cues of other species that are in the same prey guild. Rainbow Darters (Etheostoma caeruleum) are benthic fish that co‐occur with native Ozark Minnows (Notropis nubilus), recently introduced Western Mosquitofish (Gambusia affinis), and native Oklahoma Salamanders (Eurycea tyrnerensis), all of whom are vulnerable to the same predators. We tested the responses of darters to the damage‐released alarm cues of conspecifics (positive control), minnows, and mosquitofish; alarm cues from Bumblebee Gobies (Brachygobius doriae) served as a negative (allopatric) control. We also tested the response of sympatric and allopatric darters to the damage‐released alarm cues of Oklahoma Salamander. Darters exhibited a fright response to conspecific and minnow alarm cues, but not to cues from mosquitofish or gobies. Lack of response to mosquitofish cues could be because they are introduced or because they typically occur higher in the water column than darters. Darters that were sympatric with the salamander exhibited a fright response to the alarm cues of the salamander, while allopatric darters did not. Rainbow Darters can develop responses to the alarm cues of syntopic species (minnows and Oklahoma Salamander) within their prey guild.     

Identifying female phenotypes that promote behavioral isolation in a sexually dimorphic species of fish Etheostoma zonale

In sexually dimorphic species characterized by exaggerated male ornamentation, behavioral isolation is often attributed to female preferences for conspecific male signals. Yet, in a number of sexually dimorphic species, male mate choice also results in behavioral isolation. In many of these cases, the female traits that mediate species boundaries are unclear. Females in sexually dimorphic species typically lack many of the elaborate traits that are present in males and that are often used for taxonomic classification of species. In a diverse and largely sexually dimorphic group of fishes called darters (Percidae: Etheostoma), male mate choice contributes to behavioral isolation between a number of species; however, studies addressing which female traits males prefer are lacking. In this study, we identified the dominant female pattern for two sympatric species, Etheostoma zonale and Etheostoma barrenense, using pattern energy analysis, and we used discriminate function analysis to identify which aspects of female patterning can reliably classify species. We then tested the role of female features in male mate choice for E. zonale, by measuring male preference for computer animations displaying the identified (species-specific) conspecific features. We found that the region above the lateral line is important in mediating male mate preferences, with males spending a significantly greater proportion of time with animations exhibiting conspecific female patterning in this region than with animations exhibiting heterospecific female patterning. Our results suggest that the aspects of female phenotypes that are the target of male mate choice are different from the conspicuous male phenotypes that traditionally characterize 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.     

Reinforcement in the banded darter Etheostoma zonale: The effect of sex and sympatry on preferences

Reinforcement occurs when selection against hybrid offspring strengthens behavioral isolation between parental species and may be an important factor in speciation. Theoretical models and experimental evidence indicate that both female and male preferences can be strengthened upon secondary contact via reinforcement. However, the question remains whether this process is more likely to affect the preferences of one sex or the other. Males of polygynous species are often predicted to exhibit weaker preferences than females, potentially limiting the ability for reinforcement to shape male preferences. Yet, in darters (Percidae: Etheostoma), male preference for conspecific mates appears to arise before female preferences during the early stages of allopatric speciation, and research suggests that male, but not female, preferences become reinforced upon secondary contact. In the current study, we aimed to determine whether the geographically widespread darter species Etheostoma zonale exhibits a signature of reinforcement, by comparing the strength of preference for conspecific mates between populations that are sympatric and allopatric with respect to a close congener, E. barrenense. We examined the strength of preference for conspecifics for males and females separately to determine whether the preferences of one or both sexes have been strengthened by reinforcement. Our results show that both sexes of E. zonale from sympatric populations exhibit stronger conspecific preferences than E. zonale from allopatric populations, but that female preferences appear to be more strongly reinforced than male preferences. Results therefore suggest that reinforcement of female preferences may promote behavioral isolation upon secondary contact, even in a genus that is characterized by pervasive male mate choice.     

Preference for conspecifics evolves earlier in males than females in a sexually dimorphic radiation of fishes

Speciation by sexual selection is generally modeled as the coevolution of female preferences and elaborate male ornaments leading to behavioral (sexual) reproductive isolation. One prediction of these models is that female preference for conspecific males should evolve earlier than male preference for conspecific females in sexually dimorphic species with male ornaments. We tested that prediction in darters, a diverse group of freshwater fishes with sexually dimorphic ornamentation. Focusing on the earliest stages of divergence, we tested preference for conspecific mates in males and females of seven closely related species pairs. Contrary to expectation, male preference for conspecific females was significantly greater than female preference for conspecific males. Males in four of the 14 species significantly preferred conspecific females; whereas, females in no species significantly preferred conspecific males. Relationships between the strength of preference for conspecifics and genetic distance revealed no difference in slope between males and females, but a significant difference in intercept, also suggesting that male preference evolves earlier than females’. Our results are consistent with other recent studies in darters and suggest that the coevolution of female preferences and male ornaments may not best explain the earliest stages of behavioral isolation in this lineage.     

Riverscape genetics of the orangethroat darter complex

Freshwater darters belonging to the orangethroat darter species complex, or Ceasia, are widely distributed in the Central and Southern United States, with ranges that span both glaciated and unglaciated regions. Up to 15 species have been recognized in the complex, with one, Etheostoma spectabile, having a widespread northern distribution and another, Etheostoma pulchellum, having a sizeable southern distribution. The other species in the complex have much more restricted distributions in unglaciated regions of the Central Highlands. We sampled 384 darters from 52 sites covering much of the range of Ceasia and evaluated patterns of genetic diversity, genetic structure, and pre- and post-glacial patterns of range contraction and expansion. We anticipated finding much stronger signals of genetic differentiation and diversification in unglaciated regions, given the higher species diversity and levels of endemism reported there. Surprisingly, microsatellite genotyping revealed two well-differentiated genetic clusters of E. spectabile in samples from glaciated regions, one confined to the Illinois River basin and another found in the Wabash drainage and Great Lakes tributaries. This suggests that there was expansion from two isolated glacial refugia, with little subsequent post-glacial gene flow. Fish collected from throughout the unglaciated region were less genetically differentiated. Fish assigned to Etheostoma burri and Etheostoma uniporum based on collection sites and morphological characters were not genetically differentiated from E. spectabile samples from the region. Hybridization and introgression occurring in the Central Highlands may confound genetic delineation of species in this region of high endemism and diversity.     

Female mate choice and species recognition between two closely related cichlid fish of Lake Malawi,Metriaclima estherae and M. callainos

Cichlids are one of the most diverse and colourful groups of freshwater fishes in the world. Despite much investigation, the factors that promote speciation in these fishes are still uncertain. However, previous studies suggest that sexual selection on male colour is one of the main drivers of speciation among these fishes. Metriaclima estherae is a polymorphic cichlid species from Lake Malawi, and thus provides an ideal model for the investigation of the importance of colour as a species recognition cue. M. callainos is a closely related and morphologically similar species, with male colour pattern very similar to that of M. estherae. We tested female choice by giving females of the two species a choice between conspecific and heterospecific males in the presence and absence of visual (colour) and chemical cues. The results show that females of M. callainos were able to reliably recognize conspecific males, even when colour was eliminated as a cue. However, females of M. estherae did not prefer conspecific males, although they were able to discriminate between red and blue conspecific colour morphs by using chemical cues. These results suggest that species recognition cues may differ even among closely related species of cichlid fish, and that female preferences for male coloration may be weak in certain species.     

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.     

Population and Species Divergence of Chemical Cues that Influence Male Recognition of Females in Desmognathine Salamanders

Growing evidence indicates that males may be more discriminating of mating partners than often has been assumed. In the North American Ocoee dusky salamander, Desmognathus ocoee (Plethodontidae: Desmognathinae), sexual incompatibility among conspecific populations is high in encounters staged in the laboratory, at least in part because males fail to recognize ‘other’ females as appropriate targets for courtship. I used Y-mazes to test the hypothesis that males of D. ocoee discriminate between substrate-borne chemical cues produced by ‘own’ (homotypic) and ‘other’ (heterotypic) females. Males of four populations discriminated in favor of substrates soiled by homotypic females over clean (control) substrates (expt 1), suggesting that females produce chemical cues of sociosexual significance to males. Furthermore, males from these populations discriminated in favor of substrates soiled by homotypic females vs. substrates soiled by heterotypic females (expt 2), both conspecific and heterospecific (D. carolinensis and D. orestes). Thus, differences among populations and species in female chemical cues appear to affect the chemotactic responses of males. I suggest that, together with differences in behavioral signals and responses exhibited during courtship, differences in female chemical cues likely contribute to sexual incompatibility among populations and taxa of desmognathine salamanders.     

Lack of species discrimination based on chemical cues by male sailfin mollies, <Emphasis Type="Italic">Poecilia latipinna</Emphasis>

When making mating decisions, individuals may rely on multiple cues from either the same or multiple sensory modalities. Although the use of visual cues in sexual selection is well studied, fewer studies have examined the role of chemical cues in mate choice. In addition, few studies have examined how visual and/or chemical cues affect male mating decisions. Male mate choice is important in systems where males must avoid mating with heterospecific females, as is found in a mating complex of Poecilia. Male sailfin mollies, Poecilia latipinna, are sexually parasitized by gynogenetic Amazon mollies, P. formosa. Little is known about the mechanism by which male sailfin mollies base their mating decisions. Here we tested the hypothesis that male sailfin mollies from an allopatric and a sympatric population with Amazon mollies use multiple cues to distinguish between conspecific and heterospecific females. We found that male sailfin mollies recognized the chemical cues of conspecific females, but we found no support for the hypothesis that chemical cues are by themselves sufficient for species discrimination. Lack of discrimination based on chemical cues alone may be due to the close evolutionary history between P. latipinna and P. formosa. Males from populations sympatric with Amazon mollies did not differentially associate with females of either of the two species when given access to both visual and chemical cues of the females, yet males from the allopatric population did associate more with conspecific females than with heterospecific females in the presence of both chemical and visual cues. The lack of discrimination by males from the sympatric population between conspecific and heterospecific females based on both chemical and visual cues suggests that these males require more complex combinations of cues to distinguish species, possibly due to the close relatedness of these species.     

Discrimination of conspecific sex and reproductive condition using chemical cues in axolotls (<Emphasis Type="Italic">Ambystoma mexicanum</Emphasis>)

Chemosensory cues play an important role in the daily lives of salamanders, mediating foraging, conspecific recognition, and territorial advertising. We investigated the behavioral effects of conspecific whole-body odorants in axolotls, Ambystoma mexicanum, a salamander species that is fully aquatic. We found that males increased general activity when exposed to female odorants, but that activity levels in females were not affected by conspecific odorants. Although males showed no difference in courtship displays across testing conditions, females performed courtship displays only in response to male odorants. We also found that electro-olfactogram responses from the olfactory and vomeronasal epithelia were larger in response to whole-body odorants from the opposite sex than from the same sex. In males, odorants from gravid and recently spawned females evoked different electro-olfactogram responses at some locations in the olfactory and vomeronasal epithelia; in general, however, few consistent differences between the olfactory and vomeronasal epithelia were observed. Finally, post hoc analyses indicate that experience with opposite-sex conspecifics affects some behavioral and electrophysiological responses. Overall, our data indicate that chemical cues from conspecifics affect general activity and courtship behavior in axolotls, and that both the olfactory and vomeronasal systems may be involved in discriminating the sex and reproductive condition of conspecifics.Abbreviations EOG electro-olfactogram - VNO vomeronasal organ     

Analysis of Early Embryogenesis in Rainbow and Banded Darters (Percidae: Etheostoma) Reveals Asymmetric Postmating Barrier

This study examines early embryogenesis in two species of darters, Etheostoma caeruleum and E. zonale (Teleostei: Percidae), and their hybrids. Results document the course of ontogeny from fertilization until the onset of pigmented eyes. Comparing fertilization and developmental success of conspecific versus heterospecific crosses revealed an asymmetric postmating reproductive barrier. E. caeruleum eggs treated with E. zonale sperm exhibited fertilization and developmental success similar to that of conspecific crosses. In contrast, E. zonale eggs treated with E. caeruleum sperm exhibited reduced fertilization relative to conspecific crosses and abnormal development. Development in this latter cross was compromised at all stages, but appeared to be concentrated around epiboly, or cell migration. As epiboly represents the stage of ontogeny when zygotic genes of both species jointly contribute to embryogenesis, results provide insight into the genetic mechanisms underlying postmating barriers in Etheostoma. Finally, the observed asymmetry in fertilization success is consistent with predictions based on the behavioral ecology of these species. Etheostoma zonale spawn in the open water column, whereas E.␣caeruleum bury their eggs under the substrate. The observed fertilization barrier may have therefore resulted from selection favoring increased fertilization specificity in E. zonale.     

Opercular beat rate for rainbow darters Etheostoma caeruleum exposed to chemical stimuli from conspecific and heterospecific fishes

Opercular beat rates of rainbow darters Etheostoma caeruleum were used as a measure of a physiological response to chemical stimulation. Rainbow darters responded significantly to some chemical cues (active and ambush predators, competitors, novel stimuli and to conspecific and heterospecific alarm cues) with increases in opercular movements; neutral cues and novel alarm cues did not elicit significant changes. Changes in opercular movements may be a good bioassay for determining detection of chemical stimuli by rainbow darters.     

Incomplete behavioural isolation and asymmetric female preference in darter sister species (Percidae: Etheostoma)

In this study, female preferences and behavioural isolation were estimated in a pair of allopatric sister species, Etheostoma duryi and Etheostoma flavum. Dichotomous mate preference trials were conducted to determine whether females prefer to associate with conspecific over heterospecific males and free‐spawning assays were conducted to determine whether those preferences translated into behavioural isolation. Dichotomous mate choice trials revealed asymmetric female preference, as female E. flavum preferred conspecific males, whereas female E. duryi showed no preference. Free‐spawning assays indicated that behavioural isolation remains incomplete between E. duryi and E. flavum (I_B = 0·19). In addition to female mating preferences, male behaviour also appeared to influence mating outcomes as male E. flavum consistently courted conspecific females more often in free‐spawning assays whereas male E. duryi did not. The data therefore suggest that despite marked divergence in male nuptial colour, divergence in female preferences between these species may not be sufficient to maintain species boundaries upon secondary contact. These results contrast with similar work in a sympatric darter species pair and may be explained by considering the contributions of reinforcement and differences in colour pattern as well as colour value.