Mechanism For Species Recognition Research Articles

Serum species identification using mid-infrared and far-infrared spectroscopy combined with neural network algorithms

Serum is a widely used biological fluid containing rich biological information, commonly employed in clinical diagnosis and medical treatment. However, since human serum contains genetic information of races and involves national information security, accurate identification of species serum is required for customs import and export trade. In this study, we propose a species serum identification method combining mid-infrared and far-infrared spectroscopy with neural network algorithms. By collecting spectra of 147 serum samples from 8 species and conducting preliminary analysis on specific spectral ranges, differences between spectra of certain species were identified. Subsequently, a Convolutional Neural Network (CNN) model was constructed for species identification and classification, achieving recognition accuracy of 95.00 % for human and non-human serums (binary classification) and 8 species of serums (octal classification). Through correlation analysis, the corresponding relationships between species serums and their spectral characteristic regions, as well as relevant molecular groups and chemical bonds, were clarified. This reveals the underlying mechanism for species recognition based on serum spectra. Furthermore, by nesting multiple segmented progressive sub-models and optimizing the weight ratios of spectral features, the accuracy of species recognition was improved to 98.95 % while the computational complexity was greatly reduced. These results provide a reliable and efficient method for rapid differentiation of species serums, with important implications for the identification of other biological fluids.

Analysing the information content of the multimodal courtship display of a parasitoid wasp

Abstract Courtship displays are generally composed of multiple signal components that are transmitted and perceived through different sensory modalities. However, previous studies on multimodal courtship displays have focused mainly on individual signals, thus failing to provide a full explanation of why these displays evolved despite the added costs involved. Therefore, it is essential to identify the purpose of each display component and link them to prevailing hypotheses on multimodal display function. Here, we study the multimodal courtship display of males of the parasitoid wasp Leptopilina heterotoma, which consists of wing fanning and a putative antennal pheromone, by determining the information conveyed about male quality through these two modalities. We found that a high wing-fanning frequency and a large body size led to a higher mating success, whereas we found no correlation of the pheromone composition with mating success. We conclude that the wing-fanning component conveys mate-assessment information, whereas the chemical display component, found to be species specific in a previous study, functions as a species-recognition mechanism but does not reflect the quality of a male. Altogether, our study suggests that the multimodal courtship display of L. heterotoma fits the multiple message hypothesis and might thus be a useful model for further studies.

Effect of soil nutrients, neighbor identities and root separation types on intra‐ and interspecific interaction among three clonal plant species

In natural environments, plants frequently interact with both heterospecific and conspecific neighbors. The intensity of belowground plant interaction with neighboring species commonly varies with the availability of soil nutrients in the habitats. According to classical ecological theory, competition between conspecific neighbors may be more severe than competition between unrelated species due to the similar nutrient requirements of close relatives, especially when nutrients are scarce in the habitat. However, many recent studies have shown the opposite pattern, and suggested an alternative mechanism based on species recognition. Taking Zoysia sinica as the focal species, we conducted a controlled experiment to test the results of intraspecific and interspecific interactions among three clonal species Zoysia sinica, Zoysia japonica and Alternanthera philoxeroides, which represent a conspecific, a close relative and a distant relative of the focal species, respectively, and at different root treatments (no separation NS, clone separation CS and ramet separation RS) and two nutrient levels. The results showed that Z. sinica recognized conspecific plants in the NS and CS treatments, and did not show above or belowground competition with these. The performance of the focal plant (Z. sinica) was better when it was grown with a conspecific neighbor as compared to all other types of neighbors. In all root separation treatments, the competition was more intense when Z. sinica grew with a close relative (Z. japonica) than when growing with a distant relative (A. philoxeroides). Generally, competition between plants was more intense at the high nutrient level than at the low nutrient level, suggesting that both soil nutrients and a species recognition mechanism play a significant role for the intra‐ and interspecific interaction and fitness of these three neighboring clonal species.

The Role of Multimodal Signals in Species Recognition Between Tree-Killing Bark Beetles in a Narrow Sympatric Zone.

When related species coexist, selection pressure should favor evolution of species recognition mechanisms to prevent interspecific pairing and wasteful reproductive encounters. We investigated the potential role of pheromone and acoustic signals in species recognition between two species of tree-killing bark beetles, the southern pine beetle, Dendroctonus frontalis Zimmermann, and the western pine beetle, Dendroctonus brevicomis LeConte, in a narrow zone of sympatry, using reciprocal pairing experiments. Given the choice of adjacent con- or heterospecific female gallery entrance in a log, at least 85% of walking males chose the entrance of the conspecific, and half the males that initially entered heterospecific galleries re-emerged and entered the conspecific gallery within 15 min. Waveform analysis of female acoustic "chirps" indicated interspecific differences in chirp timing. Males may use information from female acoustic signals to decide whether to enter or remain in the gallery. Individuals in forced heterospecific pairings (produced by confinement of a heterospecific male within the female entrance) did not differ in pheromone production from individuals of conspecific pairs. However, due to the absence of the right species of male, galleries with heterospecific pairs released an abnormal pheromone blend that lacked at least one key component of the aggregation pheromone of either species. The complete aggregation pheromone (i.e., the pheromone blend from entrances with pairs) does not appear to deter interspecific encounters or confer premating reproductive isolation per se; however, it may confer selective pressure for the maintenance of other reproductive isolation mechanisms.

The evolution of contact calls in isolated and overlapping populations of two white-eye congeners in East Africa (Aves, Zosterops)

BackgroundClosely related species often occur in geographic isolation, yet sometimes form contact zones with the potential to hybridize. Pre-zygotic barriers may prevent cross breeding in such contact zones. In East Africa, White-eye birds have evolved into various species, inhabiting different habitat types. Zosterops poliogaster is found in cool and moist cloud forests at higher elevations, whereas Z. abyssinicus is distributed across the dry and hot lowland savannahs. In most areas, these two species occur allopatrically, but in the contact zone where the mountain meets the savannah, the distributions of these species sometimes overlap (parapatry), and in a few areas the two taxa occur sympatrically. Acoustic communication is thought to be an important species recognition mechanism in birds and an effective prezygotic barrier for hybridisation. We recorded contact calls of both the lowland and highland species in (i) distinct populations (allopatry), (ii) along contact zones (parapatry), and (iii) in overlapping populations (sympatry) to test for species and population differentiation.ResultsWe found significant differences in call characteristics between the highland and lowland species, in addition to call differentiation within species. The highland Z. poliogaster shows a strong call differentiation among local populations, accompanied by comparatively low variability in their contact calls within populations (i.e. a small acoustic space). In contrast, calls of the lowland Z. abyssinicus are not differentiated among local sites but show relatively high variability in calls within single populations. Call patterns in both species show geographic clines in relation to latitude and longitude. Calls from parapatric populations from both species showed greater similarity to the other taxon in comparison to heterospecific populations found in allopatry. However, where the two species occur sympatrically, contact calls of both species are more distinct from each other than in either allopatric or parapatric populations.ConclusionThe contrasting patterns reflect divergent spatial distributions: the highland Z. poliogaster populations are highly disjunct, while Z. abyssinicus lowland populations are interconnected. Higher similarity in contact calls of heterospecific populations might be due to intermixing. In contrast, sympatric populations show reproductive character displacement which leads to strongly divergent call patterns.

Opportunist slave-making ants Myrmoxenus ravouxi discriminate different host species from a non-host species

Slave-making ants exploit the worker force of host colonies permanently and have to make recurrent raids in order to replenish the slave’s stock. Some of these parasite species exploit different host species and few studies so far have been devoted to host species recognition mechanisms. Here, we tried to determine if opportunist slave-making ants using different host species rely on innate or experience-induced preferences to discriminate host from non-host species. We show that Myrmoxenus ravouxi slave-making workers are not only more aggressive toward heterocolonial host and potential host species workers when compared with non-host species workers, but also toward heterocolonial host workers than toward heterocolonial conspecifics. Moreover, M. ravouxi workers display more antennations and contacts toward the heterocolonial host species when compared with the non-host species. We also show that they do not discriminate between homocolonial and heterocolonial conspecifics. Together, our results suggest that this opportunistic slave-making ant species may have a complex social recognition template based on both innate and experience-based mechanisms.

Possible molecular mechanisms of species recognition by barnacle larvae inferred from multi-specific sequencing analysis of proteinaceous settlement-inducing pheromone

Gregarious settlement is essential for reproduction and survival of many barnacles. A glycoprotein, settlement-inducing protein complex (SIPC) has been recognized as a signal for settlement and it is expressed in both conspecific adults and larvae. Although the settlement-inducing activities of SIPC are species-specific, the molecular-based mechanism by which larvae distinguish conspecific SIPC from the SIPC of other species is still unknown. Here, the complete primary structure of the SIPC of Megabalanus coccopoma, as well as the partial structure of the SIPCs of Balanus improvisus, Megabalanus rosa, and Elminius modestus are reported. These SIPCs contain highly variable regions that possibly modulate the affinity for the receptor, resulting in the species specificity of SIPC. In addition, the distribution patterns of potential N-glycosylation sites were seen to be different among the various species. Differences in such post-translational modifications may contribute to the species specificity of SIPC.

The role of androgens in species recognition and sperm production in Atlantic mollies (Poecilia mexicana)

Much is known about the role of hormones in the regulation of vertebrate mating behavior, including receptivity, and several components of mate choice. Hormones may modulate reproductive behavior in such a way to increase or decrease the individual's motivation, and therefore hormones may be important in mediating behavior associated with reproductive isolation. The mating complex of the all female gynogenetic Amazon mollies, Poecilia formosa, and their parental species (sailfin mollies, P. latipinna, and Atlantic mollies, P. mexicana) is a model system for studying ultimate mechanisms of species recognition. However, proximate mechanisms, such as variation in hormone levels, and the effect of hormones on sperm production have not been extensively examined. We predict that one or more of the sex steroid hormones in teleost fish (11-ketotestosterone (KT), testosterone (T), and estradiol (E)) will play a role in species recognition (during mate choice and/or sperm priming) for Atlantic mollies (the maternal parental species) that are sympatric with Amazon mollies. We sequentially paired male Atlantic mollies with female conspecifics and Amazon mollies and obtained water-borne hormone samples before and after mating for all fish. We measured circulating KT, T, and E from the water samples. Although we did not find an overall KT response to mating with conspecifics as has been found previously in sailfin mollies, male Atlantic mollies that mated more with conspecific females had lower postmating T levels. Additionally, males attempted to mate more with conspecific females that had lower postmating E levels, but attempted to mate more with Amazon mollies that had higher postmating KT levels. We also examined the effect of KT on sperm priming (a mechanism of premating mate choice), and found that KT levels of male Atlantic mollies prior to mating are correlated with the sperm priming response when males were paired with conspecific females, but this correlation was not found when males were paired with Amazon mollies. Our results indicate that male mating behavior is affecting or responding to both male and female hormones, but that the hormones alone are not playing a role in species recognition. Male Atlantic mollies may not discriminate against Amazon mollies as strongly as male sailfin mollies because Amazon mollies resemble their maternal parental species more than their paternal species.

Species Limits in Antbirds (Thamnophilidae): The Scale-Backed Antbird (Willisornis poecilinotus) Complex

The geographic range of the Scale-backed Antbird (Willisornis poecilinotus) encompasses Amazonia. Seven currently defined subspecies are distinguished from one another by diagnostic plumage characters except for one pair. Six pairs of subspecies are apparently parapatric and lack a known barrier to intergradation in at least a portion of their contact zone; yet confirmed hybrids are known only for one pair in one location. An analysis of >350 recordings, however, found vocal differences among them insufficient to recommend elevating subspecies to the species level with one exception. Populations in southeastern Amazonia should be considered a distinct species, Willisornis vidua (Hellmayr), Xingu Scale-backed Antbird, on the basis of their distinct loudsongs, raspy call series, and contact calls. Within the widespread Willisornis poecilinotus, Common Scale-backed Antbird, the remaining instances of parapatry without extensive intergradation provide a focus for future fieldwork to define interrelationships in contact zones and mechanisms of species recognition that may be sustaining them on independent evolutionary paths.

A potential role of male and female androgen in species recognition in a unisexual–bisexual mating complex

Hormones play a critical role in the regulation of vertebrate mating behavior, including receptivity, and several components of mate choice. However, less is known about the role of these chemical messengers in mediating behavior associated with premating reproductive isolation. The bisexual–unisexual mating complex of sailfin mollies, Poecilia latipinna, and Amazon mollies, Poecilia formosa (sexual parasites of sailfins) has been a model system for studying ultimate mechanisms of species recognition. However proximate mechanisms, such as variation in hormone levels, have not been examined. We paired male sailfin mollies with either female conspecifics or Amazon mollies and obtained water-borne hormone samples before and after mating for all fish. We measured 11-ketotestosterone, testosterone, and estradiol from the water samples. As expected from previous studies, males mated with conspecifics more frequently than with Amazon mollies. 11-Ketotestosterone production by males increased when they mated with female sailfin mollies who themselves also showed elevated production of 11-ketotestosterone. This increase in male and female 11-ketotestosterone levels was not seen when males mated with Amazon mollies. This unique endocrine interaction represents a potential proximate mechanism for species recognition by male sailfin mollies. We found no significant change in testosterone or estradiol under these conditions suggesting that a single hormone mediates bidirectional interactions between males and females during courtship.

Chemosensory species recognition may reduce the frequency of hybridization between native and introduced lizards

The introduction of alien species to islands by human activity can cause catastrophic consequences for small populations of island endemics. Hybridization between the endangered and endemic insular lizard Podarcis atrata (Boscá, 1916) from the Columbretes Islands (Mediterranean, Spain) and the common mainland lizard Podarcis hispanica (Steindachner, 1870) could potentially occur because mainland haplotypes have already been detected in the islands, the two species are closely genetically related, and the frequency of visitors to these islands is increasing. However, reproductive decisions of lizards are often mediated by species recognition mechanisms based on chemical cues. On the basis of this observation, even if some mainland P. hispanica lizards were introduced to the islands, interspecific recognition might make rare an eventual hybridization with the insular P. atrata. We examined interspecific chemical recognition between the insular P. atrata and the mainland P. hispanica. Our results showed that lizards of both sexes responded more strongly (i.e., directed a significantly higher number of tongue flicks) to scents of conspecific individuals than to scents of heterospecifics. Chemical recognition of conspecifics by endemic island P. atrata lizards may reduce the occurrence of hybridization with introduced mainland P. hispanica lizards and protect the insular gene pool.

“Singing on the Wing” as a Mechanism for Species Recognition in the Malarial Mosquito Anopheles gambiae

Anopheles gambiae, responsible for the majority of malaria deaths annually, is a complex of seven species and several chromosomal/molecular forms. The complexity of malaria epidemiology and control is due in part to An. gambiae's remarkable genetic plasticity, enabling its adaptation to a range of human-influenced habitats. This leads to rapid ecological speciation when reproductive isolation mechanisms develop [1-6]. Although reproductive isolation is essential for speciation, little is known about how it occurs in sympatric populations of incipient species [2]. We show that in such a population of "M" and "S" molecular forms, a novel mechanism of sexual recognition (male-female flight-tone matching [7-9]) also confers the capability of mate recognition, an essential precursor to assortative mating; frequency matching occurs more consistently in same-form pairs than in mixed-form pairs (p = 0.001). [corrected] Furthermore, the key to frequency matching is "difference tones" produced in the nonlinear vibrations of the antenna by the combined flight tones of a pair of mosquitoes and detected by the Johnston's organ. By altering their wing-beat frequencies to minimize these difference tones, mosquitoes can match flight-tone harmonic frequencies above their auditory range. This is the first description of close-range mating interactions in incipient An. gambiae species.

A test for conspecific cueing in two sympatric species of pupfish (Cyprinodon beltrani, C. simus)

In many fishes, individuals prefer to associate with phenotypically similar or conspecific individuals (conspecific cueing). Such phenotypic segregation can be an important evolutionary driver, for example, in intralacustric sympatric speciation processes. I examined conspecific cueing in two species of sympatric pupfish from Laguna Chichancanab in southern Mexico: the little shoaling and highly territorial Cyprinodon beltrani and the highly shoaling but non-territorial C. simus. Females were tested for shoal species preferences in two testing scenarios: (1) a sequential choice test where shoals of four conspecific or four heterospecific (Cyprinodon sp. or Poecilia reticulata) females were presented in succession, and (2) a simultaneous choice test where female shoals of both Cyprindon species were presented concurrently. Overall, higher shoaling in C. simus was corroborated in this study. In the sequential test, no effect of the type of stimulus shoal (con- or heterospecific) on shoaling behavior was detected. In the simultaneous tests, C. beltrani, but not C. simus females showed a preference for the conspecific shoal. It seems possible that C. simus females did not evolve species recognition mechanisms because no other Cyprinodon species in the Laguna Chichancanab shows equally high shoaling, which automatically leads to the formation of single-species (i.e., C. simus-) shoals. C. simus males do not establish long-term territories, but rather spawn within shoals, whereas C. beltrani females approach males in their breeding territories to spawn. I discuss that this behavioral difference still provides a powerful reproductive isolation mechanism even in the absence of conspecific cueing in C. simus.

Effect of conductivity changes on the stability of electric signal waveforms in dwarf stonebashers (Mormyridae; Pollimyrus castelnaui, P. marianne)

The dwarf stonebasher sibling species Pollimyrus castelnaui and P. marianne use differences in the electric organ discharges (EODs) for species recognition. As EOD waveforms are affected by water conductivity changes, the reliability of species recognition might be impeded due to natural variability in the environment. EODs of P. castelnaui (N = 8) and P. marianne (N = 8) under high (250 microS/cm) and low (25 microS/cm) conductivity were recorded and compared. Local peaks of the EODs of both species were significantly and predictably modified due to the conductivity change but species-specific differences were always recognizable. The duration of the EODs was not influenced by the conductivity change. Temperature alterations modified the duration in a linear relationship, allowing the determination of Q(10) values (1.6 for P. castelnaui's and 1.7 for P. marianne's EODs). As the species-specific differences are not masked by conductivity effects, EOD discrimination seems to be a reliable species recognition mechanism under natural circumstances.

The structure of sulfated polysaccharides ensures a carbohydrate-based mechanism for species recognition during sea urchin fertilization

The evolution of barriers to inter-specific hybridization is a crucial step in the fertilization of free spawning marine invertebrates. In sea urchins, molecular recognition between sperm and egg ensures species recognition. Here we review the sulfated polysaccharide-based mechanism of sperm-egg recognition in this model organism. The jelly surrounding sea urchin eggs is not a simple accessory structure; it is molecularly complex and intimately involved in gamete recognition. It contains sulfated polysaccharides, sialoglycans and peptides. The sulfated polysaccharides have unique structures, composed of repetitive units of alpha-L-fucose or alpha-L-galactose, which differ among species in the sulfation pattern and/or the position of the glycosidic linkage. The egg jelly sulfated polysaccharides show species-specificity in inducing the sperm acrosome reaction, which is regulated by the structure of the saccharide chain and its sulfation pattern. Other components of the egg jelly do not possess acrosome reaction inducing activity, but sialoglycans act in synergy with the sulfated polysaccharide, potentiating its activity. The system we describe establishes a new view of cell-cell interaction in the sea urchin model system. Here, structural changes in egg jelly polysaccharides modulate cell-cell recognition and species-specificity leading to exocytosis of the acrosome. Therefore, sulfated polysaccharides, in addition to their known functions as growth factors, coagulation factors and selectin binding partners, also function in fertilization. The differentiation of these molecules may play a role in sea urchin speciation.

Female mate choice determines reproductive isolation between sympatric butterflies

Animal courtship rituals are important for species recognition, and a variety of cues might be utilized to recognize conspecific mates. In this paper, we investigate different species-recognition mechanisms between two sympatric butterfly sister species: the wood white (Leptidea sinapis) and Real’s wood white (Leptidea reali). We show that males of both species frequently court heterospecific females both under laboratory and field conditions. The long-lasting elaborate courtships impose energetic costs, since the second courtship of males that were introduced to two subsequent conspecific females lasted on average only one fourth as long as the first courtship. In this paper, we demonstrate that premating reproductive isolation is dependent on female unwillingness to accept heterospecific mates. We studied female and male courtship behavior, chemical signaling, and the morphology of the sexually dimorphic antennae, one of the few male traits visible for females during courtship. We found no differences in ultraviolet (UV) reflectance and only small differences in longer wavelengths and brightness, significant between-species differences, but strongly overlapping distributions of male L. sinapis and L. reali antennal morphology and chemical signals and minor differences in courtship behavior. The lack of clear-cut between-species differences further explains the lack of male species recognition, and the overall similarity might have caused the long-lasting elaborate courtships, if females need prolonged male courtships to distinguish between con- and heterospecific suitors.

Hybridization between two sympatrically breeding species of fur seal at Iles Crozet revealed by genetic analysis

Introgressive hybridization occurs when closely related taxa overlap in distribution and is often associated with historically isolated populations coming into contact as a result of anthropogenic disturbance. There is evolutionary and conservation interest in detecting hybridization to determine its implications on future species composition, especially for threatened and recovering taxa such as subantarctic (Arctocephalus tropicalis) and Antarctic (A. gazella) fur seals, which were driven to the brink of extinction by human exploitation. Hybridization between these species has been reported at two locations and they breed sympatrically at a third site, Iles Crozet. While hybrid individuals have previously been identified based on phenotype, individuals can be difficult to classify based on these characteristics alone. Genotypic hybrid identification has been successful in several species, including fur seals. In this study we conducted an assignment test using microsatellite data to identify hybrids and to measure the frequency of hybridization at Iles Crozet. Samples were collected from 372 individuals and screened with 6 polymorphic microsatellite markers. MtDNA genotypes were also determined for individuals identified as hybrids or backcrosses based on microsatellite genotype. Phenotype, microsatellite and mtDNA genotype were then compared in order to identify hybrids. The results indicate that 1% of the population have hybrid genotypes and at a minimum, 2.4% of the population are backcrossed to parental species. We found that the two species are genetically distinct from one another and given the low rate of hybridization it is unlikely that they will fuse. These results suggest that there is a mechanism for species recognition that acts as a barrier to hybridization. It therefore seems unlikely that fur seals are threatened by significant introgression. Further investigation of fur seal mating systems would provide valuable insight into the mechanisms governing hybridization and species recognition in mate choice.

Dispersive and non-dispersive waves through plants: implications for arthropod vibratory communication

Vibratory communication in arthropods is a widespread phenomenon. Arthropods living on plants have been reported to use only dispersive bending waves in the context of prey-predator, competition, social and sexual interactions. Differences in signal structure have also been postulated to work as species recognition mechanisms and speciation agents. Using two identical laser Doppler vibrometers and a wavelet analysis, we quantified the wave propagation modes in rush stems (Juncus effusus) over the whole range of frequencies used by arthropods. A non-dimensionalized analysis shows that mechanical waves propagate not only as dispersive bending waves, but also as non-dispersive waves. Our analysis implies that an arthropod can communicate through non-dispersive bending waves by either producing signals of high frequencies or by choosing large stems, two widely different options tapping into the physiological and the behavioural repertoires, respectively. Non-dispersive waves, unreported so far in insect vibratory communication in plants, present serious advantages over dispersive bending waves in terms of signal integrity and may well be much more widely used than anticipated, in particular for species recognition.

Reduction of the association preference for conspecifics in cave-dwelling Atlantic mollies, Poecilia mexicana

Cave animals are widely recognised as model organisms to study regressive evolutionary processes like the reduction of eyes. In this paper, we report on the regressive evolution of species discrimination in the cave molly, Poecilia mexicana, which, unlike other cave fishes, still has functional eyes. This allowed us to examine the response to both visual and non-visual cues involved in species discrimination. When surface-dwelling females were given a chance to associate with either a conspecific or a swordtail (Xiphophorus hellerii) female, they strongly preferred the conspecific female both when multiple cues and when solely visual cues were available to the female. No association preference was observed when only non-visual cues were provided. In contrast, cave-dwelling females showed no preference under all testing conditions, suggesting that species recognition mechanisms have been reduced. We discuss the role of species discrimination in relation to habitat differences.

Auditory matching of male Epipedobates femoralis (Anura: Dendrobatidae) under field conditions

In the evolution of species recognition mechanisms, the differential reactions of receivers towards a signal may be as important as variation in the signal features. In many animals, the auditory system's most sensitive range often matches the dominant frequency range of the signals. The degree of spectral matching, however, can be modified by stimulus intensity and, therefore, patterns detected in the laboratory should ideally be confirmed by field experiments. Territorial males of the dart-poison frog, Epipedobates femoralis , approach vocally active conspecific intruders. Since phonotaxis (followed by fighting) affects male fitness, the auditory system is predicted to discriminate against heterospecific calls. We tested the rôle of spectral and temporal call features in call discrimination by conducting playback experiments in the field with synthetic calls. To study the effect of heterospecific interference on the male's phonotactic reaction, we measured signal parameters and signalling activity of four sympatric species. The highest probability of response coincided with the spectral and temporal features of the population's advertisement call and the probability of approach was matched to the average frequency of this call. In addition, the degree of matching was sensitive to the number of notes per call. The decrease in the probability of response, however, was steeper at lower than average frequencies, where the probability of heterospecific interference was highest. Our results suggest that under field conditions frequency tuning interacts with temporal call features in maximizing the detection of conspecific signals and reducing the probability of heterospecific interference.