Song Phenotype Research Articles

Genomic regions underlying the species-specific mating songs of green lacewings.

Rapid species radiations provide insight into the process of speciation and diversification. The radiation of Chrysoperla carnea-group lacewings seems to be driven, at least in part, by their species-specific pre-mating vibrational duets. We associated genetic markers from across the genome with courtship song period in the offspring of a laboratory cross between Chrysoperla plorabunda and Chrysoperla adamsi, two species primarily differentiated by their mating songs. Two genomic regions were strongly associated with the song period phenotype. Large regions of chromosomes one and two were associated with song phenotype, as fewer recombination events occurred on these chromosomes relative to the other autosomes. Candidate genes were identified by functional annotation of proteins from the C. carnea reference genome. The majority of genes that are associated with vibrational courtship signals in other insects were found within QTL for lacewing song phenotype. Together these findings suggest that decreased recombination may be acting to keep together loci important to reproductive isolation between these species. Using wild-caught individuals from both species, we identified signals of genomic divergence across the genome. We identified several candidate genes both in song-associated regions and near divergence outliers including nonA, fruitless, paralytic, period, and doublesex. Together these findings bring us one step closer to identifying the genomic basis of a mating song trait critical to the maintenance of species boundaries in green lacewings.

The puzzling, rarely encountered European lacewings Chrysoperla ankylopteryformis and C. renoni (Neuroptera: Chrysopidae): one species or two?

ABSTRACT Two of the most intriguing yet poorly understood taxa in the Chrysoperla carnea-group of green lacewings are the European species (alternatively, forms or ecotypes) known as Chrysoperla renoni and C. ankylopteryformis. These two rare taxa have been variously combined and separated by systematists over the years, but their taxonomic status remains controversial, partly because the two have very unusual characteristics, and partly because they are simply difficult to find in nature. Here, we introduce new observations and experimental results, gathered from living individuals collected at new localities in Hungary (C. renoni) and Spain (C. ankylopteryformis). These data encompass duetting song phenotype, mate choice and species recognition, body colour, pretarsal claw dimensions, wing setal length, and predominant habitat associations. The inheritance of several of these traits is also explored, by hybridising each of the two focal taxa with their abundant, co-occurring relative in the carnea-group, C. pallida. Our findings support recognition of C. renoni and C. ankylopteryformis each as valid, independent species, thereby also raising the latter species from its current synonymy with C. renoni (formally, Chrysoperla ankylopteryformis Monserrat & Díaz-Aranda, 1989 stat. rev.). We propose an evolutionary hypothesis to explain the reproductive isolation of these species from one another, as well as from C. pallida. https://www.zoobank.org/urn:lsid:zoobank.org:pub:7800468B-0E79-4634-B523-FDF18BC3EC95

Like Father Like Son: Cultural and Genetic Contributions to Song Inheritance in an Estrildid Finch.

Social learning of vocalizations is integral to song inheritance in oscine passerines. However, other factors, such as genetic inheritance and the developmental environment, can also influence song phenotype. The relative contributions of these factors can have a strong influence on song evolution and may affect important evolutionary processes such as speciation. However, relative contributions are well-described only for a few species and are likely to vary with taxonomy. Using archived song data, we examined patterns of song inheritance in a domestic population of Java sparrows (Lonchura oryzivora), some of which had been cross-fostered. Six-hundred and seventy-six songs from 73 birds were segmented and classified into notes and note subtypes (N = 22,972), for which a range of acoustic features were measured. Overall, we found strong evidence for cultural inheritance of song structure and of the acoustic characteristics of notes; sons’ song syntax and note composition were similar to that of their social fathers and were not influenced by genetic relatedness. For vocal consistency of note subtypes, a measure of vocal performance, there was no apparent evidence of social or genetic inheritance, but both age and developmental environment influenced consistency. These findings suggest that high learning fidelity of song material, i.e., song structure and note characteristics, could allow novel variants to be preserved and accumulate over generations, with implications for evolution and conservation. However, differences in vocal performance do not show strong links to cultural inheritance, instead potentially serving as condition dependent signals.

Urban sparrows respond to a sexually selected trait with increased aggression in noise

Animals modify acoustic communication signals in response to noise pollution, but consequences of these modifications are unknown. Vocalizations that transmit best in noise may not be those that best signal male quality, leading to potential conflict between selection pressures. For example, slow paced, narrow bandwidth songs transmit better in noise but are less effective in mate choice and competition than fast paced, wide bandwidth songs. We test the hypothesis that noise affects response to song pace and bandwidth in the context of competition using white-crowned sparrows (Zonotrichia leucophrys). We measure male response to song variation along a gradient of ambient noise levels in San Francisco, CA. We find that males discriminate between wide and narrow bandwidth songs but not between slow and fast paced songs. These findings are biologically relevant because songs in noisy areas tend to have narrow bandwidths. Therefore, this song phenotype potentially increases transmission distance in noise, but elicits weaker responses from competitors. Further, we find that males respond more strongly to stimuli in noisier conditions, supporting the ‘urban anger’ hypothesis. We suggest that noise affects male responsiveness to song, possibly leading to more territorial conflict in urban areas.

Behavioural response to song and genetic divergence in two subspecies of white-crowned sparrows (Zonotrichia leucophrys).

Divergence in sexual signals may drive reproductive isolation between lineages, but behavioural barriers can weaken in contact zones. Here, we investigate the role of song as a behavioural and genetic barrier in a contact zone between two subspecies of white-crowned sparrows (Zonotrichia leucophrys). We employed a reduced genomic data set to assess population structure and infer the history underlying divergence, gene flow and hybridization. We also measured divergence in song and tested behavioural responses to song using playback experiments within and outside the contact zone. We found that the subspecies form distinct genetic clusters, and demographic inference supported a model of secondary contact. Song phenotype, particularly length of the first note (a whistle), was a significant predictor of genetic subspecies identity and genetic distance along the hybrid zone, suggesting a close link between song and genetic divergence in this system. Individuals from both parental and admixed localities responded significantly more strongly to their own song than to the other subspecies song, supporting song as a behavioural barrier. Putative parental and admixed individuals were not significantly different in their strength of discrimination between own and other songs; however, individuals from admixed localities tended to discriminate less strongly, and this difference in discrimination strength was explained by song dissimilarity as well as genetic distance. Therefore, we find that song acts as a reproductive isolating mechanism that is potentially weakening in a contact zone between the subspecies. Our findings also support the hypothesis that intraspecific song variation can reduce gene flow between populations.

Natural courtship song variation caused by an intronic retroelement in an ion channel gene.

Animal species display enormous variation for innate behaviours, but little is known about how this diversity arose. Here, using an unbiased genetic approach, we map a courtship song difference between wild isolates of Drosophila simulans and Drosophila mauritiana to a 966 base pair region within the slowpoke (slo) locus, which encodes a calcium-activated potassium channel. Using the reciprocal hemizygosity test, we confirm that slo is the causal locus and resolve the causal mutation to the evolutionarily recent insertion of a retroelement in a slo intron within D. simulans. Targeted deletion of this retroelement reverts the song phenotype and alters slo splicing. Like many ion channel genes, slo is expressed widely in the nervous system and influences a variety of behaviours; slo-null males sing little song with severely disrupted features. By contrast, the natural variant of slo alters a specific component of courtship song, illustrating that regulatory evolution of a highly pleiotropic ion channel gene can cause modular changes in behaviour.

Patterns of Song across Natural and Anthropogenic Soundscapes Suggest That White-Crowned Sparrows Minimize Acoustic Masking and Maximize Signal Content.

Soundscapes pose both evolutionarily recent and long-standing sources of selection on acoustic communication. We currently know more about the impact of evolutionarily recent human-generated noise on communication than we do about how natural sounds such as pounding surf have shaped communication signals over evolutionary time. Based on signal detection theory, we hypothesized that acoustic phenotypes will vary with both anthropogenic and natural background noise levels and that similar mechanisms of cultural evolution and/or behavioral flexibility may underlie this variation. We studied song characteristics of white-crowned sparrows (Zonotrichia leucophrys nuttalli) across a noise gradient that includes both anthropogenic and natural sources of noise in San Francisco and Marin counties, California, USA. Both anthropogenic and natural soundscapes contain high amplitude low frequency noise (traffic or surf, respectively), so we predicted that birds would produce songs with higher minimum frequencies in areas with higher amplitude background noise to avoid auditory masking. We also anticipated that song minimum frequencies would be higher than the projected lower frequency limit of hearing based on site-specific masking profiles. Background noise was a strong predictor of song minimum frequency, both within a local noise gradient of three urban sites with the same song dialect and cultural evolutionary history, and across the regional noise gradient, which encompasses 11 urban and rural sites, several dialects, and several anthropogenic and natural sources of noise. Among rural sites alone, background noise tended to predict song minimum frequency, indicating that urban sites were not solely responsible for driving the regional pattern. These findings support the hypothesis that songs vary with local and regional soundscapes regardless of the source of noise. Song minimum frequency from five core study sites was also higher than the lower frequency limit of hearing at each site, further supporting the hypothesis that songs vary to transmit through noise in local soundscapes. Minimum frequencies leveled off at noisier sites, suggesting that minimum frequencies are constrained to an upper limit, possibly to retain the information content of wider bandwidths. We found evidence that site noise was a better predictor of song minimum frequency than territory noise in both anthropogenic and natural soundscapes, suggesting that cultural evolution rather than immediate behavioral flexibility is responsible for local song variation. Taken together, these results indicate that soundscapes shape song phenotype across both evolutionarily recent and long-standing soundscapes.

Parallel evolution in courtship songs of North American and European green lacewings (Neuroptera: Chrysopidae)

Green lacewings of the Chrysoperla carnea species group use substrate-borne vibrational duetting songs rather than morphology or pheromones for species recognition. Because each of the many cryptic, reproductively isolated song species typically has an extensive geographic range, potentially interfertile biological species are broadly sympatric, and therefore must use distinct regions of acoustic song space if they are to remain reproductively isolated. However, this constraint does not apply to species restricted to different continents, giving rise to the possibility of parallel evolution of song phenotypes between continents. Here we describe a striking example of parallel song evolution, between a known European species, Chrysoperla pallida, and a newly discovered vivid-green North American species, Chrysoperla calocedrii sp. nov. To verify this parallelism, we show that: (1) the songs of the two species have measurably similar multi-volley temporal and frequency structure; (ii) the songs share the same basic genetic pathway; (iii) each species is unable to discriminate between its own and the other's song in playback trials, confirming the acoustic niche overlap; (iv) the two species readily establish normal duets with each other in the lab, leading to copulation and the production of vigorous hybrid progeny bearing an intermediate song phenotype; (v) they have distinct morphologies in both adults and larvae, suggesting different adaptive responses and therefore independent evolutionary histories; and (vi) they occupy relatively distant positions in a Bayesian phylogenetic analysis of 4630 bp of protein-coding mitochondrial DNA, rejecting the alternative hypothesis of similarity through recent common ancestry. We include a formal description of C. calocedrii sp. nov. as a new species, and provide additional observations of its behaviour, ecology, and life history. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105, 776–796.

Acoustic niche partitioning in two cryptic sibling species of Chrysoperla green lacewings that must duet before mating

Cryptic species may seem identical to us, but not to each other. The Chrysoperla carnea group of green lacewings has species-specific substrate-borne duetting songs that maintain reproductive isolation among its cryptic species. We propose that sympatric species of the group will partition acoustic niche space so as to minimize mating mistakes. Sibling species pairs could therefore occupy abutting regions of acoustic space without hybridizing in nature. One species pair, Chrysoperla plorabunda and C. adamsi, are not only closest relatives, but are also the most acoustically similar among sympatric congeners. Using F1 hybrids as a surrogate taxon occupying acoustic space between the parents, we tested the hypothesis that this space cannot support an additional species. Focusing on a critical song feature, volley period, we confirm firstly that the hybrid song occupies unfilled signal space between the parents. Secondly, we show that song discrimination is strong between parents, but weaker between parents and hybrids. Thirdly, each parent has the ability to respond to the other’s signal in a narrow region of overlap. Finally, preference functions for hybrids broadly overlap those of parents. These acoustic tests confirm that a species of this intermediate song phenotype could not persist.

Testing the developmental stress hypothesis in canaries: consequences of nutritional stress on adult song phenotype and mate attractiveness

The complex songs of songbirds are thought to have evolved through sexual selection. Sexually selected signals must be associated with costs in order to ensure their honesty as indicator of male quality. Costs may relate to the development of the neural substrate underlying song learning, which develops already very early in life. Song may, therefore, serve as an indicator of the early developmental history. This nutritional stress hypothesis has initially been confirmed for a variety of species, but recent studies using zebra finches as a model species reported somewhat inconsistent effects, and the functional consequences of changes in adult song phenotype remain unclear. We tested the nutritional stress hypothesis in canaries by manipulating either the brood size or the food quality postfledging. The brood size manipulation had a significant effect on early development, and low food quality postfledging led to a transient reduction in body mass. However, we did not find evidence that any of the song traits measured reflected the early developmental conditions, which is in conflict with the nutritional stress hypothesis. Canaries may be less vulnerable to nutritional stress or are able to compensate stressful conditions during early development. However, if males compensated, this compensation may have come at a survival cost. Female mate choice decisions were independent of the developmental history of a male. Instead, females preferred males singing longer song bouts, a trait that may contain a heritable component.

Mitochondrial Phylogeny of Cryptic Species of the Lacewing Chrysoperla nipponensis (Neuroptera: Chrysopidae) in Japan

Abstract We surveyed a 1,033-bp fragment of the cytochrome oxidase subunit I (COI) sequence to look for a useful diagnostic marker for discrimination between the Japanese cryptic species Chrysoperla nipponensis (Okamoto) (Neuroptera: Chrysopidae) (types A and B) and the commercially introduced Chrysoperla carnea s. str. from Germany. Phylogenetic analysis showed four distinct haplotype groups: one group corresponded to C. nipponensis type B and one group to C. carnea s. str., and the remaining two distinct groups, A1 and A2, corresponded to the same song phenotype, C. nipponensis type A. A2 was linked with the group of C. carnea s. str. and A1 was linked with the group of C. nipponensis type B. Cross-testing between A1 and A2 revealed their reproductive compatibility. Mitochondrial polyphyly within C. nipponensis type A may, therefore, originate from introgression or incomplete lineage sorting. Within the mitochondrial COI region presented here, there were 17 variable nucleotide sites useful for discriminating the four haplotype groups from each other.

Elevated yolk androgen levels and the expression of multiple sexually selected male characters

Maternal hormones in bird eggs modulate not only offspring development, but recently it has also been shown that these effects can persist into adult life. A number of long-lasting effects concern traits of which the expression or development is modulated by androgens. This suggests that the nature of yolk hormone-mediated maternal effects may be organizational. Maternal androgens may therefore play an important role in sexual selection, since the expression of sexually selected male characters is often androgen-dependent. We experimentally manipulated the yolk androgen concentrations of European Starling ( Sturnus vulgaris) eggs. Subsequently we followed 49 unrelated males from hatching until year of first reproduction. We investigated the expression of multiple sexually selected male characters (song, beak color and throat feather characteristics), taking into account whether a trait is androgen-dependent. Elevated levels of yolk androgens affected the length of the embryonic period, but did not modify the expression of either androgen-dependent or androgen-independent sexually selected male characters including song phenotype at adulthood. Thus the most important function of yolk androgens in starlings and possibly other bird species may relate to the early developmental period. The outcome of our study together with the results of our meta-analysis indicates that the effects of yolk androgens on sexually selected male characters may be comparatively small. Our results suggest that this may relate to the numerous other environmental and/or genetic factors influencing the expression of sexually selected male characters.

Yolk testosterone, postnatal growth and song in male canaries

Avian eggs contain substantial amounts of maternal yolk androgens, which have been shown to modulate offspring phenotype. The first studies on the functional consequences of maternal yolk androgens have focused on early life stages and their role in sibling competition. However, recent longitudinal studies reported long-lasting effects of maternal yolk androgens on offspring phenotype, mostly concerning traits that are sensitive to androgens. This suggests that maternal yolk androgens could play an important role in sexual selection, since the expression of many male sexual characters is testosterone-dependent. Using male canaries as a model, we examined the consequences of an experimental elevation of yolk testosterone concentrations on early development as well as long-lasting effects particularly on song, which is one of the most important sexual characters in male songbirds. Elevated yolk testosterone concentrations inhibited male growth, possibly in interaction with an existent ectoparasite exposure. Males hatched from testosterone-treated eggs (T-males) did not have enhanced competitive skills, in contrast to previous studies. The elevation of yolk testosterone concentrations delayed song development but did not affect adult song phenotype. This is intriguing, as yolk testosterone possibly induced developmental stress, which is known to reduce song quality. We hypothesize that yolk testosterone has either no direct effect on adult song phenotype, or that positive effects are merged by the negative effects of developmental stress. Finally, females mated with T-males invested more in their clutch indicating that females either assess T-males as more attractive (differential allocation hypothesis) or compensated for lower offspring viability (compensation hypothesis).

Adaptation or random change? The evolutionary response of songs to substrate properties in lacewings (Neuroptera: Chrysopidae: Chrysoperla)

Green lacewings of the carnea group of Chrysoperla constitute a large complex of cryptic species that differ principally in their substrate-borne, vibrational mating signals. Sexual selection has very likely contributed to their recent and rapid divergence. Some theoretical models require that evolutionary environmental adaptation assist sexual selection during speciation, whereas others do not. Here, we looked for evidence of adaptation having shaped the song phenotypes of lacewing species that have different habitat preferences. First, we looked for a pattern of convergent song evolution, predicted under a hypothesis of substrate adaptation during speciation. Focusing on species confined to conifers versus those on herbaceous vegetation, we found several examples where song phenotype showed a correlation with substrate that was independent of phylogeny. We also present results of experimental tests of environmental adaptation using C. downesi and C. plorabunda , which are conifer and meadow species, respectively. Experiments addressing purely biomechanical aspects of substrate transmission showed that the songs of C. downesi and C. plorabunda propagated equally well in either grass stems or conifer branches. Behavioural experiments showed, furthermore, that individuals did not discriminate between conspecific songs played through the ‘correct’ versus the ‘incorrect’ substrate. We conclude that the pattern suggestive of environmental adaptation of lacewing songs to their substrates was not supported by experiments that explicitly tested for adaptation. The carnea group of cryptic species may be one of the few examples where sexual selection has acted on arbitrary differences in premating signals, resulting in speciation.

Hormone-dependent neural plasticity in the juvenile and adult song system: what makes a successful male?

The sexual quality of adult song is the result of genetic and epigenetic mechanisms shaping the neural song system throughout life. Genetic brain-intrinsic mechanisms determine the neuron pools that develop into forebrain song control areas independent of gonadal steroid hormones, androgens and estrogens. One fate of these neurons is the potential to express sex steroid receptors, such as androgen and estrogen receptors. Genetic brain-intrinsic mechanisms, too, determine the activity of hypothalamic-pituitary-gonad (HPG) axis, i.e., the working range and responsiveness of HPG axis to produce gonadal hormones. The epigenetic action of gonadal steroid hormones (androgens and estrogens) on determined vocal neurons is required to maintain and increase the pool of determined vocal neurons and to complete the connections of the vocal system, i.e., to make it function motorically. The subsequent influence of environmental information, including both external (socio-sexual and physical) and internal (body physiology) signals, specify the further neural phenotype of vocal areas either through acting on the HPG axis and differential release of gonadal hormones or through non-gonadal hormone systems, both of which have target neurons in the functional vocal system. Despite the clear evidence of hormone dependency of the development of both the adult song phenotype and song system phenotype, their causal relation is complex.

A lacewing with the wanderlust: the European song species ‘Maltese’, Chrysoperla agilis, sp.n., of the carnea group of Chrysoperla (Neuroptera: Chrysopidae)

Abstract. A fifth European member of the Holarctic carnea sibling species group, Chrysoperla agilis, sp.n. is described. This species was initially recognized because of its unique song phenotype. Both sexes produce one type of song, characterized by a single very long volley averaging 4.5 s in duration and repeated every 9.5 s. The unit of exchange during heterosexual duets (the shortest repeated unit) is the single volley. The carrier frequency is upwardly, then downwardly, modulated during each volley. The maximum frequency is higher (around 110 Hz) than in other members of the carnea group. Each volley ends with an audible, percussive ‘rattle’. Song features vary only slightly over a large geographical range that extends from northern Iran westward to the Azores and northward to southern Switzerland. Discriminant function analysis confirms the uniqueness of the C. agilis song in comparison with nine other song species. Morphologically, the adult of C. agilis is very similar to C. carnea and C. pallida and is generally intermediate between them with respect to the size of the pretarsal basal dilation, the shape of the genital lip and chin, the colour and type of setae on the abdomen, and colour markings on the head and mouthparts. No single feature will distinguish C. agilis from either C. carnea or C. pallida, but a suite of physical attributes can be used with reasonable success. Larval morphology fails to distinguish clearly C. agilis from other song species. Ecologically, C. agilis shares an affinity for low vegetation with C. lucasina and C. carnea, and a preference for milder, Mediterranean climates with C. mediterranea. Adults undergo a colour change to pale yellowish brown during winter diapause, and eggs are laid singly rather than in groups. Based on its ecological characteristics, C. agilis may prove to be an effective agent of biological control in the agricultural fields of warmer, drier parts of southern Europe and the Middle East, where other members of the carnea group are less common or absent.

FEMALE LIFE SPAN AND FERTILITY ARE INCREASED BY THE EJACULATES OF PREFERRED MALES

In animals with internal fertilization, sperm competition among males can favor the evolution of male ejaculate traits that are detrimental to females. Female mating preferences, in contrast, often favor traits in males that are beneficial to females, yet little is known about the effect of these preferences on the evolution of male ejaculates. A necessary condition for female preferences to affect the evolution of male ejaculate characteristics is that females select mates based on a trait correlated with ejaculate quality. Previous work has shown that females of the variable field cricket, Gryllus lineaticeps, prefer males that produce calling songs containing faster and longer chirps. In this study, we tested the hypothesis that females receive more beneficial ejaculates from preferred males. Females were placed on either a high- or a reduced-nutrition diet then mated twice to a male of known song phenotype. Females received only sperm and seminal fluid from males during these matings. There was no effect of male song phenotype on any fitness component for females on the high-nutrition diet. Reduced-nutrition females mated to males that produced preferred song types, however, lived longer, produced more eggs, produced more fertile eggs, and had a higher proportion of their eggs fertilized than those mated to other males. The life-span benefit was positively associated with male chirp duration, and the reproductive benefits were positively associated with male chirp rate. We explored two possible mechanisms for the life span and reproductive benefits. First, a path analysis suggested that part of the effect of male chirp duration on female life span may have been indirect; females mated to males that produced longer chirps showed delayed oviposition, and females that delayed oviposition lived longer. Males that produce longer chirps may thus transfer fewer or less potent oviposition stimulants to females in their seminal fluid. Second, there was a positive correlation between male chirp rate and the number of sperm transferred to females. The fertility benefit may thus have resulted from females receiving more sperm from males that produce faster chirps. Finally, there was a negative phenotypic correlation between male chirp rate and chirp duration, suggesting that females may have to trade off the life span and reproduction benefits when selecting a mate.

Molecular Dissection of the 5′ Region of no-on-transientA of Drosophila melanogaster Reveals cis-Regulation by Adjacent dGpi1 Sequences

The nonA gene of Drosophila melanogaster is important for normal vision, courtship song, and viability and lies approximately 350 bp downstream of the dGpi1 gene. Full rescue of nonA mutant phenotypes can be achieved by transformation with a genomic clone that carries approximately 2 kb of 5' regulatory material and that encodes most of the coding sequence of dGpi1. We have analyzed this 5' region by making a series of deleted fragments, fusing them to yeast GAL4 sequences, and driving UAS-nonA expression in a mutant nonA background. Regions that both silence and enhance developmental tissue-specific expression of nonA and that are necessary for generating optomotor visual responses are identified. Some of these overlap the dGpi1 sequences, revealing cis-regulation by neighboring gene sequences. The largest 5' fragment was unable to rescue the normal electroretinogram (ERG) consistently, and no rescue at all was observed for the courtship song phenotype. We suggest that sequences within the nonA introns that were missing in the UAS-nonA cDNA may carry enhancer elements for these two phenotypes. Finally, we speculate on the striking observation that some of the cis-regulatory regions of nonA appear to be embedded within the coding regions of dGpi1.

Revised concept of Chrysoperla mediterranea (Hölzel), a green lacewing associated with conifers: courtship songs across 2800 kilometres of Europe (Neuroptera: Chrysopidae)

SummaryThe Holarctic carnea‐group of Chrysoperla green lacewings consists of numerous cryptic, sibling species, distinguished principally by vibrational courtship songs. A European member of this species‐group, C. mediterranea (Hölzel), was originally diagnosed by its dark green coloration, red band across the front of the head, narrow wings, minute basal dilation of the pretarsus and ecological association with pines. Here we describe its courtship and mating song, which uniquely distinguishes the species from all other members of the carnea‐group in Europe. Males and females both produce a single type of song, characterized by a long train of short, closely spaced volleys and nearly constant carrier frequency. Measured song features are nearly invariant across a large geographical range that extends well outside that previously known for C. mediterranea. Principal components analysis confirms the uniqueness of the mediterranea song in comparison with five other sympatric song species of the carnea‐group. Populations which show the mediterranea song phenotype do not necessarily retain all of the traditional morphological features of the species, particularly in eastern parts of the range. Specifically, the green coloration may be less intense, the wings broader and the pretarsal basal dilation larger, resulting in significant morphological overlap with other song species. Larval morphology and ecophysiology also fail to distinguish C. mediterranea from other song species. Based on unique song phenotype, however, we conclude that C. mediterranea is a valid biological species, which can also be recognized in museum collections when a suite of physical attributes is used.

GENETIC VARIATION IN CRICKET CALLING SONG ACROSS A HYBRID ZONE BETWEEN TWO SIBLING SPECIES.

The sibling ground crickets Allonemobius fasciatus and A. socius meet along a mosaic hybrid zone at ≈ 40°N latitude in eastern North America. In this paper we report the findings of a genetic analysis of calling-song variation within and among six cricket populations sampled along a transect through the hybrid zone in southern New Jersey. We compared aspects of the calling song of both wild-caught and laboratory-reared crickets to test the hypothesis that population differences in song observed in the wild were genetically based. We found significant, species-level differences in all aspects of the calling song, and these differences persisted even after a generation of common-garden rearing in the laboratory, supporting the hypothesis that interspecific variation observed in the wild largely reflects genetic differentiation between the two taxa. A discriminant function analysis indicated that individual crickets could be assigned to the proper taxon with less than 10% error, supporting the premise that calling song could be used by female crickets as a mechanism for species recognition. One population, collected from within the hybrid zone and containing significant numbers of hybrid individuals, was intermediate in its calling song, presumably reflecting this population's mixed genetic makeup. In this hybrid zone population, song phenotype was highly correlated to a hybrid index score generated using species-specific alleles at four diagnostic allozyme markers, suggesting a multigenic basis to calling-song variation in these crickets as well as linkage disequilibrium between markers and song. Based on an analysis of laboratory-reared full-sib families, broad-sense heritabilities for calling-song characteristics were generally significant in the two A. socius populations, whereas many components of song showed no significant family effects in the three A. fasciatus populations. The genotypically mixed, hybrid zone population showed very high heritabilities for most calling-song components, which likely reflect the influence of interspecific gene flow on genetic variation for quantitative traits.