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Singing and cryptic speciation insects
REVIEWS
Singingand cryptic s
ion in insects
Charles S. Henry
for speciation through song diBiologists have long known of the ’nsect songs have always invergence. existence of ‘inaudible’ songs in insects, trigued us. In simpler times, but recent work has shown such naturalists often anthropo1 morphized the chirpings, trill- substrate-borne or near-field signals to be General theoretical background virtually ubiquitous, and often correlated ings, raspings and buzzings of the Many theories have been with high numbers of sibling species. familiar insect singers. Singing or advanced to explain how evolchorusing individuals were happy In a sexual context, silent singing has the ution might proceed and how formidable advantage of privacy: neither and exuberant, angry or frightpredators nor sexual competitors can species might arise from the conened, lusty or jealous, or perhaps tinued divergence of sexually listen and disrupt. Privacy enhances just complaining or bragging about selected traits, such as songs. their circumstances. Later, evolspecies recognition by promoting signal utionary thinking infiltrated hySeveral related models from complexity. It also encourages the population genetics emphasize evolution of intricate signals in females potheses of insect song function, as well as males, leading to obligatory producing a mechanistic view of runaway correlated selection on dueting behavior during pair formation. acoustical signaling, and sophistithe male signal system and the cated electronic tools were deCurrent evidence suggests that song female response systems-5. Under divergence in dueting taxa can facilitate veloped to test the predictions of such models, traditional adaprapid, sympatric speciation. those theories. However, accepted tation plays little role: arbitrary interpretations of the 1960s and song features become established early 1970s have been displaced in subpopulations through the Charles Henry is at the Dept of Ecology & in recent years by alternative exvagaries of female choice, accelplanations, developed through rig- Evolutionary Biology, University of Connecticut, U-43, erated by an evolutionary feed75 North Eagleville Road, Storrs, CT 06269, USA. orous application of individual back loop. Eventually, full species selection models to sexual behavstatus is achieved, but only if the ior. In particular, views favoring species discrimination specific prerequisites of each model are met. Unforand reproductive isolation as primary functions of songs tunately, it is difficult to formulate predictions that will have given way to those emphasizing intrasexual distinguish one model from another unambiguously”. competition or individual assessment of mate quality’. Another problem concerns the interpretation of female Ironically, Darwin had argued years earlier that the comresponses to male signals. In typical acoustical insects, plex temporal structures of insect songs had been promales sing and females mutely orient to the singers; conseduced like any other exaggerated feature, through female quently, it is difficult to design experiments that accuchoice and sexual selection, so in a sense we have come rately assess the female’s often subtle reaction to a male’s full circle in our thinking. call. Thus, song-mediated speciation of this type is plausible, Sexually selected traits have been implicated in the probut unsupported by convincing examples. A second set of models approaches the issue of species cess of speciation, so presumably songs can also promote speciation. Certainly, different song types are strongly as- origin through song from a traditional ecological perspective, citing reinforcement as the key process in the diversociated with biological species units in many insect groups. gence of acoustical signals during speciation2. It is assumed Furthermore, previously unrecognized, reproductively iso that divergence of populations begins for other reasons, lated sister species have been discovered in almost every for example a geographical barrier, or differences in habiacoustical taxon that has been studied in detai12.However, tat selection or host preference. Songs will change later, it is not clear whether changes in songs precede speciation, to increase assortative mating and thereby cut off harminduce speciation, or develop as a consequence of other ful gene flow between the incipient species. This process evolutionary processes in populations that have already predicts more-pronounced song differences in sympatric speciated. than in allopatric populations of closely related species. The role of songs in speciation may be easier to docuUnfortunately, conditions for the operation of true rement in a second major category of insect singers - those inforcement are thought to be very restrictive - perhaps that produce complex, inaudible songs during courtship prohibitively so?. A related process, reproductive character and mating. Until recently, such insects were thought to displacement, achieves the same final result as reinforceconstitute a small minority of taxa, but now we appreciate ment, but through selection against wasted reproductive that they are simply more difficult to recognize. Most are small, inconspicuous insects with low intensity, ‘near-field’ effort between species already establisheda. Reproductive character displacement, then, has no relevance to specior substrate-borne songs that can be detected only with ation. Many putative examples that have been used to supspecial equipment. In contrast to audible insects, silent singers typically show much less sexual dimorphism of sig- port speciation by reinforcement are better interpreted as cases of reproductive character displacement. Among nals and morphology, and usually establish duets during these we must include most of the cases of enhanced courtship. For several such silent taxa, evidence is accumulating to support a causal association between song evol- song differences observed in closely related, sympatric species of drosophilid and tephritid flies and acoustical ution and speciation. In fact, the very cases where classiOrthoptera9,‘“. cal sexual selection is weakest provide the best support
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0 1994. Elsevier Science
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Silent songs The models above are Table 1. Partial taxonomic distribution of inaudible sexual songs in Insecta not very satisfactory, but Order/Suborder Family are particularly weak when Mechanism Signal type 2 Song Duetrng ____ applied to insects with inOrthoptera Tettigoniidae tremulation abdomen yes yes audible courtship songs. 7 Gryllidae drumming yes leg Such insects produce com7 wings Phalangopsrdae air vortices yes plex, stereotyped signals, Psocoptera Psocidae yes drumming abdomen ? but exhibit few of the behavSternorrhyncha Psyllidae vrbratron abdomen? ? yes ioral patterns characteristic Auchenorrhyncha Delphacidae tymbal vibratton yes yes of sexually selected species. Cixiidae vibratron tymbal yes yes Little intrasexual (male-male) Meenoplidae ? vibratron tymbal yes Cercopidae vibration tymbal ? competition is evident, and yes vibratron tymbal Membracidae yes sexual dimorphism is miniyes Cicadellidae yes vtbratron tymbal yes mal. Both sexes of a given Heteroptera yes Gerridae H >O waves body yes species sing similarly and Pentatomrdae vibratron tymbal-like ? yes actively choose their mates, Cydnidae tymbal-like vibration yes yes based upon what they hear. vrbratron tymbal-like Reduvirdae ? yes Closely related, sympatric Plecoptera (widespread) drumming abdomen yes yes species have very different abdomen tremulation Megaloptera Sialidae ? yes songs, but inter-specific difabdomen Raphidioptera Raphidiidae tremulation yes yes ferences are usually neither tremulabon abdomen lnocelliidae yes yes abdomen Neuroptera Chrysopidae tremulatron more nor less pronounced yes yes 3 abdomen ? tremulation Hemerobiidae in populations of those same head ? drummrng Coleoptera Anobiidae yes species that happen to be elytral ? ? strrdulation Scarabaeidae allopatric. ? elytral ? stridulation Bruchidae Silent singers have elytral ? stridulation Scolflidae yes been described from Hemipelytral no stridulation Curculionidae yes tera, Orthoptera. Diptera, abdomen tremulation Mecoptera Panorpidae yes yes Plecoptera, Coleoptera and wings; near-field: Drosophilidae Drptera yes yes Neuroptera, but their actual vibration abdomen near-field wings taxonomic distribution is Tephritidae yes yes yes abdomen tremulation Chloropidae greater probably much yes 7 leg-thorax ? stridulation (several familres) Siphonaptera (Table 1). Within such taxa, songs may play a unique and profound role in the Special biological properties of silent songs process of speciation, resulting in the formation of Because they are inherently short-range and private, swarms of sibling species distinguished nearly exclusively silent songs are much less likely than audible ones to beby arbitrary, non-adaptive differences in their courtship come associated with mate attraction or intrasexual rivalry. signals. They are also more likely to evolve in females as well as males, because the risk of detection by enemies is Iowls. The physical nature of silent songs The physics of vibrating structures dictate that air- Thus, it seems that minimal sexual dimorphism, female singing, heterosexual courtship dueting and absence of overt borne sound will be expensive to produce: transferring intermale competition for females are all directly favored vibrational energy from a solid object to a gas at audible by the evolution of private acoustical communication sysfrequencies is an extremely inefficient process. The probtems. Both males and females can then use their songs to lem is exacerbated in small organisms like insects, which assess characteristics of their partners; because both sexes possess miniature acoustic radiators. It is no accident are exercising choice, there will exist a high degree of that the best known insect singers are large-bodied species discrimination through behavior alone. species, for which the high energetic cost of broadcasting loud signals is relatively less than for smaller insectsrl. If partners must exchange precise acoustical signals in a Other, ‘silent’ modes of communication will therefore be duet before they can mate, then particularly strong specific favored in small insects (Fig. 1). One type is near-field mate recognition systems16 will become established in each species. The properties of the mate recognition system can air pulsing, where individuals in very close proximity use their wings as pistons to produce low-amplitude puffs be arbitrary, just as in a conventional sexually selected of air delivered in complex temporal sequence@. Another trait, and the strength of reproductive isolation will have type is the direct transmission of vibrational signals little to do with how long the species have been separated through the substrate that connects two insects. Here, or how distinct they are morphologically or genetically. How can changes in an all-important mate recognition the signal usually results from the insect drumming system result in speciation? If genetic control of song phenoor buzzing some appropriate body part against the surtype is relatively simple, then a single mutation could proface upon which it is standing’“. Alternatively, the insect will shake some portion of its body to produce a signal, duce a new song type that is unacceptable to members of without striking the substrate]“. These are private the parental population. A single serendipitous success in mating by an individual bearing the mutation is required to courtship songs, in which the advantages of long-distance introduce the new gene into the populationIT. Thereafter, advertisement are sacrificed for freedom from interference by potential predators, parasites and conspecific intergenetic recombination and strong assortative mating based lopers. on song type could soon establish two distinctive
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Fig. 1. Three methods by which small insects produce inaudible courtship signaW. (a) Many Diptera produce complex patterns of pulsed air by flickingtheir wings in close proximity to their partners. These low-frequency signals (100-500 Hz) are in the form of particle displacement waves, which are intense and highly directional, but travel less than 10 mm from the source. (b) Most of the smaller auchenorrhynchan Hemiptera excite the substrate by vibration of paired tymbals, located dorsally at the base of the abdomen in both males and females. These organs resemble the tymbals of male cicadas, but are not backed by large resonant tracheal air sacs and therefore cannot produce audible sounds. Energy is concentrated below 1 kHz, and is transmitted to the substrate through the legs or mouthpart stylets. (c) Green lacewings (Chrysoperla) jerk or oscillate their abdomens vertically at 30-120 Hz, shaking the substrate sympathetically39. Extremely sensitive mechanoreceptors in the tibiae of the legs are tuned sharply to the frequency range characteristic of the species, Long alternating heterosexual duets must precede copulation.
genotypes, each with separate and incompatible mate recognition systems. Support for certain aspects of this model exists in real insect groups, described below. Songs of Diptera Near-field songs have been well studied in the true flies (Diptera). Wing-flicking during courtship and mating occurs in both sexes of many drosophilids and tephritids (Fig. la), producing complex patterns of pulsed air over very short distances that are decoded by the antennaels. In typical small-bodied drosophilids, female singing and heterosexual dueting are commonplace. However, male songs are more elaborate and less variable (more stereotyped) than female signals, and songs differ most markedly between adjacent and sympatric specie+‘. Furthermore, vibrational and other acoustical signaling in the larger-bodied Hawaiian Drosophila have clearly evolved under traditional sexual selection, as components of rivalry or lekking displays performed principally by males*O.Thus, drosophilids do not conform well to the speciation model for silent singers suggested above. Such a finding is not unexpected: even small mainland drosophilids use wing-flicking partly as a visual or olfactory display, and their larger Hawaiian relatives produce clearly audible clicks and hums during courtship. The patterns of recent, rapid and repeated speciation seen in several groups of Drosophila are therefore more likely to have resulted from founder-flush events (rapid evolutionary divergence resulting from the relaxation of stabilizing selection after a founder event)zl, or from bouts of intense sexual selection mediated by other types of signals. 390
A few dipterans are known to use substrate-borne vibrational signals during courtship. Hawaiian Drosophila siloestris vibrate their abdomens to produce a low-frequency purr*“, while small chloropid flies of the genus Lipara produce trains of species-specific pulses through a similar mechanism. Receptive males and females of the latter typically establish duets before mating, although the female signal differs from that of the male in several respect+. Details of species isolation in Lipara are not known, but their reproductive behavior is consistent with speciation facilitated by silent songs. Songs of Auchenorrhyncha Many families of small Auchenorrhyncha (order Hemiptera), including planthoppers, leafhoppers, treehoppers and spittlebugs, use abdominal tymbals (Fig. lb) to produce substrate-borne vibrational signals during courtship and mating24.25.As in Diptera, the silent songs of auchenorrhynchs are often found in both sexes of a species and have been implicated in reproductive isolation and speciation. Typical of the tymbal singers is the brown planthopper (Nilczpatuatu/ugens)*G.This is a single morphological species, but in Australia it is subdivided into at least two acoustically distinct biological species, each associated with a different host plant. Courting partners engage in vibrational duets, and the songs of females are very similar to those of conspecific males. In broad areas of sympatry, male and female songs are the principal barriers to hybridization between these sibling species. Because song acts as a homogametic mechanism, a series of biological species have differentiated rapidly within Nilaparuata, possibly through incidental changes in song phenotype27. This example clearly provides support for a variant of the simple speciation model outlined earlier. It is plausible that noncontinuous, arbitrary changes in songs have cut off gene flow between populations that were initially associated with different host plants. A parallel situation is seen in another planthopper, Ribautodelphaxz8. Female singing and heterosexual dueting are important components of reproductive behavior, and the female song phenotype is highly species-specific. Males appear to be even more discriminating of song parameters than are females, reversing the normal logic of female choice. DeWinterz8 takes a broad view of sexual selection, in which species recognition is considered a sub set of female and/or male choice. Male choice in Ribautodelphux may then function principally in conspecific recognition, because it is advantageous for such decisions to be made early in courtship, which incidentally places a selective premium on precise, invariant female signalsZg. Sexual selection therefore acts on both sexes in different ways: males compete among themselves for access to females, while the reduced pool of females compete intrasexually so as to assure their rapid insemination by discriminating males. The concerted effect of these pressures on the song phenotype of both sexes could conceivably drive divergence and speciation very rapidly. Studies on other genera of planthoppers confirm this pervasive pattern of host-associated sibling species distinguished principally by their songs. Among many possible examples, one can cite Jauesella30 or ProkelisW, each of which contains several closely related dueting species that have been shown to be reproductively isolated from one another, at least in part, by their complex courtship songs. As in Nilaparuata, powerful, bidirectional mate choice could accelerate the reproductive divergence of populations that were initially associated with a series of host plants, resulting in rapid and repeated speciation events.
REVIEWS One final noteworthy example is drawn from the treehoppers (Membracidae). Like the planthopper taxa above, Enchenopa bino&ta is actually a complex of sympatric biological species, each affiliated with a different host plant species. The life history of each of the nine Enchenopu species is intimately associated with the phenology of its host; consequently, accidental host shifts apparently have a strong reproductive isolating effect, which could result in sympatric speciatior+““. Very recent work, however, has documented species-specific differences in the substrateborne songs of both males and females of Enchenopa, suggesting that song divergence has also influenced the process and pattern of evolution34. Although it was originally thought that host-associated factors had driven the species apart and now kept them reproductively isolated, vibrational songs are equally effective causes of assortative mating and speciation. The mere existence of such courtship signals had not been suspected when the evolution of this species complex was first investigated. Songs of Chrysopidae (Neuroptera) Communication through the substrate is part of the ground plan of Neuroptera and its plesiomorphic sister orders, Megaloptera and Raphidioptera. Members of all three groups produce low-frequency vibrational songs by vigorously jerking (but not drumming) the abdomen (Fig. lc). The best-studied taxon is Chrysoperla, in the diverse neuropteran family Chrysopidae (green lacewings). These insects generate volleys of abdominal vibration when sexually receptive”5. The song either consists of simple trains of one volley type, or is organized further into groups of volleys of one or more different types (Fig. 2). Songs are species-specific, and both males and females sing identically, alternating single volleys or groups of volleys in precise duets during courtship and mating. The carrier frequency typically sweeps up or down in pitch during each volley. Within the genus, sexual dimorphism is minimal, and there is little evidence of intrasexual competition for mates: both sexes are equally discriminating during courtship. Also, song differences between closely related species are not greater in sympatry than they are in allopatry36. As in the hoppers, morphological species are not necessarily congruent with biological species in Chrysoperiu. For example, Chrysoperla carnea is a single morphological species, with a holarctic distribution. Morphological homogeneity across this vast geographical range applies to the finest details of male and female genitalia, and gel electrophoresis has proven no more effective than morphology for delineating taxonomic units within the species37J8. However, in recent years, well-defined ‘song morphs’ of C. curnea have been discovered, each with consistently different courtship signals and extensive distributions. Unlike hoppers, these song morphs are not characterized by different ecological niches, and often coexist at a single site, even living together on the same individual tree or bush. At least five of them have been detected in North America and five more in Europe (Fig. 3). All efforts to find hybrids between the morphs in the field have failed, and the results of playback experiments indicate that songs have an extreme isolating effect in both sympatric and allopatric population+‘. Clearly, the song morphs of C. curnea are good biological species, comprising a swarm of sibling cryptic taxa”“. To conform to our model of speciation through song divergence, genetic control of song phenotype should be relatively simple. Fortunately, the song species of the C. curneu complex hybridize in the laboratory when given
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Time (seconds) Fig. 2. Oscillograph of the complete song of Chrysoperla]ohnsonF. showing typical song features used to distinguish the biological species of the C. camea species group. (a) volley duration; (b) volley repetition interval: (c), (d) and (e) frequency (pitch) of start, middle and end of volley, respectively: (f) duration of last songvolley; (g) number of volleys per SRU (shortest repeated unit of song); (h) SRU duration.
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C. carnea ‘motorboat’
C. mediterranea
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Tme (seconds) Fig. 3. Oscillographs of the songs of nine biologtcal species of the C. camea species group, all drawn to the same scale (12 seconds). Species above the dotted line are from North America, while those below the ltne are from Europe17.
no choice, and F,, F, and backcross generations are viable and reasonably fertilel7.41.Available evidence is consistent with straightforward mendelian inheritance of song features at two or three different loci. Each species could be homozygous for a different set of alleles at each of those loci, which would produce the observed result: F, hybrids with intermediate and invariant song phenotypes [Fig. 4).
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REVIEWS A single gene mutation at a song-controlling locus would then alter singing behavior significantly, disrupting courtship between a mutant and a normal individual. A final consideration is the strength of assortative mating attributable to courtship songs. This has been tested in F, hybrids between two North American song species42, which discriminate as strongly against their parents’ songs as each of their parents do against the songs of non-conspecifics. It is therefore not so far-fetched to suggest that the gene for a new song in a population might quickly become fixed in a reproductively isolated subset of individuals possessing that song. Repeated occurrence in different regions would produce the swarms of closely related, sibling species that we see today.
General considerations I have suggested a new way of thinking about the process of speciation in insects characterized by inaudible, silent songs. The special acoustical and genetic characteristics of some of these insects can produce nearly instantaneous reproductive isolation of populations with new songs, unencumbered by adaptive responses to the environment, geographical isolation, reinforcement, repro ductive character displacement or genetic revolution. Unfortunately, in evolutionary biology, there is a disturbing tendency for each taxon to respond to its own set of rules, and it may be that Chrysoperla green lacewings are stolidly unique. Nonetheless, the data from other silently singing Diptera and Auchenorrhyncha summarized earlier
(a)
are suggestive of alternative modes of speciation in insects
possessing vibrational or near-field courtship signals. A useful approach for the future is to assess the phylogenetic relationships among the silent singers in a given taxon, using independent criteria (e.g. molecular markers). By de termining the directions and pattern of speciation in each group, it will be possible to choose one model over another with more confidence. Acknowledgements For help in all aspects of the lacewing work described here, I thank Phillip A. Adams, Stephen Brooks, Peter Duelli, James B. Johnson, Raymond J. Pupedis and Marta M. Wells. Julie J. Henry and Marta M. Wells critically read and improved the manuscript. The research was supported in part by grants from the National Science Foundation (DEB-9220579) and the University of Connecticut Research Foundation (FRS440393).
References 1 Alcock, J. and Gwynne, D.T.(1991)in Reproductive Behaviour of insects (Bailey, W.J. and Ridsdill-Smith, J., eds), pp. 10-41, Chapman &Hall 2 Otte, D. (1992) J Orthoptera Res. 1,25-49 3 Lande, R. (1981) Proc. Nat/Acad. Sci. USA 78,3721-3725 4 West-Eberhard, M.J. (1984) in insect Communication (Lewis, T., ed.), pp. 283-324, Academic Press 5 Kirkpatrick, M. (1987) Annu. Rev Ecol. Syst. 18,43-70 6 Houde. A.E. (1993) Am. Nat. 141, 796-803 7 Ritchie, M.G.,Butlin, R.K. and Hewitt, GM. (1992) Biol. J Linn. Sec.
45,219-234 8 Butlin, R.K. (1987) Am. Nat. 130,461-464 9 Coyne, J.A. and Orr, H.A. (1989) Evolution 43,362-381 10 Walker, T.J. (1974)Am. Zoo/. 14,1137-1150 11 Forrest, T.G. (1991) Behav. Ecol. 2,327-338 12 Bennet-Clark, H.C. (1971) Nature 234,255-259 13 Stewart, K.W., Bottorff. R.L., Knight. A.W. and Moring, J.B. (1991) Ann. Entomol. Sot. Am. 84,201-206 14 Morris, G.K. (1980) Anim. Behov. 28,42-51 15 BennetClark,H.C.,Leroy,Y.and Tsacas, L (198O)Anim. Behov. 28,230-255 16 Paterson, H.E.H.(1986) in Species and Speciotion (Transuool Museum Monograph No. 4) (Vrba, E.S., ed.), pp. 21-29, Transvaal Museum 17 Henry, C.S. (1985) Euokttion 39,965-984 18 Sivinski, J., Burk, T. and Webb, J.C. (1984)Anim. Eehao. 32, 1011-1016 19 Ewing, A.W. (1970) Rev. Comport Anim. 4,3-8 20 Hoikkala, A., Hoy, R.R. and Kaneshiro, K.Y.(1989) Anim. Behav. 37. 927-934
123456789 Song phenotype
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21 22 23 24 25 26 27
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2
-iiiilii Song phenotype Fig. 4. lnhentance
2
301 x plorabunda
x downesi
123456789 Song phenotype
of song phenotypes In F,, F, and backcross progeny of Chrysoperla plorabunda x C. downesi, two North American sibling species of the C. carnea species complex. (a) Oscillographs of the songs of each species. (b) Oscillographs and phenotype frequencies of F, hybrids. (c) Phenotype frequencies of F, hybrids. (d) Phenotype frequencies of backcross progeny. Song phenotypes are classified into nine categories, based on a suite of features. The extremes, types 1 and 9, represent the songs of parental C. plorabunda and C. downesi, respectively17.
28 29 30 31 32 33 34 35 36 37
38 39 40
Carson, H.L.and Templeton, A.R.(1984)Annu. Rev. Ecol. Syst. 1597-131 Hoy, R.R.,Hoikkala,A. and Kaneshiro, K.Y.(1988)Science 270,217-219 Kanmiya, K. (1990) J. Ethol. 8,105-l 19 Ossianilsson, F. (1949) Opusc. Entomot. Suppl. 10, 1-145 Hunt, R.E. (1993) Ann. Entomot. Sot. Am. 86,356-361 Claridge, M.F. (1990) Con. J Zool. 68, 1741-1746 Bailey, W.J. (1991) Acoustic Behoviour oflnsects: an Evolutionary Perspectiue, Chapman &Hall DeWinter, A.J. (1992) J Euo[. Biol. 5,249-265 Von Schilcher, F. and Dow, M. (1977)Zeitschr. Tierpsychoi.43,304-310 DeVrijer, P.W.F. (1986) Neth. J. Zool. 36, 168-179 Heady, S.E. and Denno, R.F. (1991) J. Insect Behav. 4,367-390 Wood, T.K. and Keese, M. (1990) Evolution 44,619-628 Wood, T.K. (1993) Annu. Rev. Entomot. 38,409-435 Hunt, R.E.J NY. Entomot. Sot. (in press) Henry, C.S. (1980)Ann. Entomot. Sot. Am. 73,617-621 Henry, C.S. and Wells, M.M.(1990)Ann. Entomot. Sot. Am. 83,317-325 Bullini, L.. Principi, M.M.and Cianchi, R. (1984) in h-ogress in World’s Neuropterotogy (Gepp, J., Asp&k, H. and Holzel, H., eds), pp. 57-59, Thalerhof Wells, M.M.Ann. Entomol. Sot. Am. (in press) Wells, M.M. and Henry, C.S. (1992) Evolurion 46,31-42 Henry, C.S., Wells, M.M.and Pupedis, R.J. (1993) Ann. Entomot. Sot. Am. 86, l-13
41 42
Wells, M.M. (1993) Can. J Zoot. 71, 233-237 Wells, M.M. and Henry, C.S. J fnsect Behau. (in press)
Singingand cryptic s
ion in insects
Charles S. Henry
for speciation through song diBiologists have long known of the ’nsect songs have always invergence. existence of ‘inaudible’ songs in insects, trigued us. In simpler times, but recent work has shown such naturalists often anthropo1 morphized the chirpings, trill- substrate-borne or near-field signals to be General theoretical background virtually ubiquitous, and often correlated ings, raspings and buzzings of the Many theories have been with high numbers of sibling species. familiar insect singers. Singing or advanced to explain how evolchorusing individuals were happy In a sexual context, silent singing has the ution might proceed and how formidable advantage of privacy: neither and exuberant, angry or frightpredators nor sexual competitors can species might arise from the conened, lusty or jealous, or perhaps tinued divergence of sexually listen and disrupt. Privacy enhances just complaining or bragging about selected traits, such as songs. their circumstances. Later, evolspecies recognition by promoting signal utionary thinking infiltrated hySeveral related models from complexity. It also encourages the population genetics emphasize evolution of intricate signals in females potheses of insect song function, as well as males, leading to obligatory producing a mechanistic view of runaway correlated selection on dueting behavior during pair formation. acoustical signaling, and sophistithe male signal system and the cated electronic tools were deCurrent evidence suggests that song female response systems-5. Under divergence in dueting taxa can facilitate veloped to test the predictions of such models, traditional adaprapid, sympatric speciation. those theories. However, accepted tation plays little role: arbitrary interpretations of the 1960s and song features become established early 1970s have been displaced in subpopulations through the Charles Henry is at the Dept of Ecology & in recent years by alternative exvagaries of female choice, accelplanations, developed through rig- Evolutionary Biology, University of Connecticut, U-43, erated by an evolutionary feed75 North Eagleville Road, Storrs, CT 06269, USA. orous application of individual back loop. Eventually, full species selection models to sexual behavstatus is achieved, but only if the ior. In particular, views favoring species discrimination specific prerequisites of each model are met. Unforand reproductive isolation as primary functions of songs tunately, it is difficult to formulate predictions that will have given way to those emphasizing intrasexual distinguish one model from another unambiguously”. competition or individual assessment of mate quality’. Another problem concerns the interpretation of female Ironically, Darwin had argued years earlier that the comresponses to male signals. In typical acoustical insects, plex temporal structures of insect songs had been promales sing and females mutely orient to the singers; conseduced like any other exaggerated feature, through female quently, it is difficult to design experiments that accuchoice and sexual selection, so in a sense we have come rately assess the female’s often subtle reaction to a male’s full circle in our thinking. call. Thus, song-mediated speciation of this type is plausible, Sexually selected traits have been implicated in the probut unsupported by convincing examples. A second set of models approaches the issue of species cess of speciation, so presumably songs can also promote speciation. Certainly, different song types are strongly as- origin through song from a traditional ecological perspective, citing reinforcement as the key process in the diversociated with biological species units in many insect groups. gence of acoustical signals during speciation2. It is assumed Furthermore, previously unrecognized, reproductively iso that divergence of populations begins for other reasons, lated sister species have been discovered in almost every for example a geographical barrier, or differences in habiacoustical taxon that has been studied in detai12.However, tat selection or host preference. Songs will change later, it is not clear whether changes in songs precede speciation, to increase assortative mating and thereby cut off harminduce speciation, or develop as a consequence of other ful gene flow between the incipient species. This process evolutionary processes in populations that have already predicts more-pronounced song differences in sympatric speciated. than in allopatric populations of closely related species. The role of songs in speciation may be easier to docuUnfortunately, conditions for the operation of true rement in a second major category of insect singers - those inforcement are thought to be very restrictive - perhaps that produce complex, inaudible songs during courtship prohibitively so?. A related process, reproductive character and mating. Until recently, such insects were thought to displacement, achieves the same final result as reinforceconstitute a small minority of taxa, but now we appreciate ment, but through selection against wasted reproductive that they are simply more difficult to recognize. Most are small, inconspicuous insects with low intensity, ‘near-field’ effort between species already establisheda. Reproductive character displacement, then, has no relevance to specior substrate-borne songs that can be detected only with ation. Many putative examples that have been used to supspecial equipment. In contrast to audible insects, silent singers typically show much less sexual dimorphism of sig- port speciation by reinforcement are better interpreted as cases of reproductive character displacement. Among nals and morphology, and usually establish duets during these we must include most of the cases of enhanced courtship. For several such silent taxa, evidence is accumulating to support a causal association between song evol- song differences observed in closely related, sympatric species of drosophilid and tephritid flies and acoustical ution and speciation. In fact, the very cases where classiOrthoptera9,‘“. cal sexual selection is weakest provide the best support
1
388
0 1994. Elsevier Science
Ltd
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REVIEWS
Silent songs The models above are Table 1. Partial taxonomic distribution of inaudible sexual songs in Insecta not very satisfactory, but Order/Suborder Family are particularly weak when Mechanism Signal type 2 Song Duetrng ____ applied to insects with inOrthoptera Tettigoniidae tremulation abdomen yes yes audible courtship songs. 7 Gryllidae drumming yes leg Such insects produce com7 wings Phalangopsrdae air vortices yes plex, stereotyped signals, Psocoptera Psocidae yes drumming abdomen ? but exhibit few of the behavSternorrhyncha Psyllidae vrbratron abdomen? ? yes ioral patterns characteristic Auchenorrhyncha Delphacidae tymbal vibratton yes yes of sexually selected species. Cixiidae vibratron tymbal yes yes Little intrasexual (male-male) Meenoplidae ? vibratron tymbal yes Cercopidae vibration tymbal ? competition is evident, and yes vibratron tymbal Membracidae yes sexual dimorphism is miniyes Cicadellidae yes vtbratron tymbal yes mal. Both sexes of a given Heteroptera yes Gerridae H >O waves body yes species sing similarly and Pentatomrdae vibratron tymbal-like ? yes actively choose their mates, Cydnidae tymbal-like vibration yes yes based upon what they hear. vrbratron tymbal-like Reduvirdae ? yes Closely related, sympatric Plecoptera (widespread) drumming abdomen yes yes species have very different abdomen tremulation Megaloptera Sialidae ? yes songs, but inter-specific difabdomen Raphidioptera Raphidiidae tremulation yes yes ferences are usually neither tremulabon abdomen lnocelliidae yes yes abdomen Neuroptera Chrysopidae tremulatron more nor less pronounced yes yes 3 abdomen ? tremulation Hemerobiidae in populations of those same head ? drummrng Coleoptera Anobiidae yes species that happen to be elytral ? ? strrdulation Scarabaeidae allopatric. ? elytral ? stridulation Bruchidae Silent singers have elytral ? stridulation Scolflidae yes been described from Hemipelytral no stridulation Curculionidae yes tera, Orthoptera. Diptera, abdomen tremulation Mecoptera Panorpidae yes yes Plecoptera, Coleoptera and wings; near-field: Drosophilidae Drptera yes yes Neuroptera, but their actual vibration abdomen near-field wings taxonomic distribution is Tephritidae yes yes yes abdomen tremulation Chloropidae greater probably much yes 7 leg-thorax ? stridulation (several familres) Siphonaptera (Table 1). Within such taxa, songs may play a unique and profound role in the Special biological properties of silent songs process of speciation, resulting in the formation of Because they are inherently short-range and private, swarms of sibling species distinguished nearly exclusively silent songs are much less likely than audible ones to beby arbitrary, non-adaptive differences in their courtship come associated with mate attraction or intrasexual rivalry. signals. They are also more likely to evolve in females as well as males, because the risk of detection by enemies is Iowls. The physical nature of silent songs The physics of vibrating structures dictate that air- Thus, it seems that minimal sexual dimorphism, female singing, heterosexual courtship dueting and absence of overt borne sound will be expensive to produce: transferring intermale competition for females are all directly favored vibrational energy from a solid object to a gas at audible by the evolution of private acoustical communication sysfrequencies is an extremely inefficient process. The probtems. Both males and females can then use their songs to lem is exacerbated in small organisms like insects, which assess characteristics of their partners; because both sexes possess miniature acoustic radiators. It is no accident are exercising choice, there will exist a high degree of that the best known insect singers are large-bodied species discrimination through behavior alone. species, for which the high energetic cost of broadcasting loud signals is relatively less than for smaller insectsrl. If partners must exchange precise acoustical signals in a Other, ‘silent’ modes of communication will therefore be duet before they can mate, then particularly strong specific favored in small insects (Fig. 1). One type is near-field mate recognition systems16 will become established in each species. The properties of the mate recognition system can air pulsing, where individuals in very close proximity use their wings as pistons to produce low-amplitude puffs be arbitrary, just as in a conventional sexually selected of air delivered in complex temporal sequence@. Another trait, and the strength of reproductive isolation will have type is the direct transmission of vibrational signals little to do with how long the species have been separated through the substrate that connects two insects. Here, or how distinct they are morphologically or genetically. How can changes in an all-important mate recognition the signal usually results from the insect drumming system result in speciation? If genetic control of song phenoor buzzing some appropriate body part against the surtype is relatively simple, then a single mutation could proface upon which it is standing’“. Alternatively, the insect will shake some portion of its body to produce a signal, duce a new song type that is unacceptable to members of without striking the substrate]“. These are private the parental population. A single serendipitous success in mating by an individual bearing the mutation is required to courtship songs, in which the advantages of long-distance introduce the new gene into the populationIT. Thereafter, advertisement are sacrificed for freedom from interference by potential predators, parasites and conspecific intergenetic recombination and strong assortative mating based lopers. on song type could soon establish two distinctive
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Fig. 1. Three methods by which small insects produce inaudible courtship signaW. (a) Many Diptera produce complex patterns of pulsed air by flickingtheir wings in close proximity to their partners. These low-frequency signals (100-500 Hz) are in the form of particle displacement waves, which are intense and highly directional, but travel less than 10 mm from the source. (b) Most of the smaller auchenorrhynchan Hemiptera excite the substrate by vibration of paired tymbals, located dorsally at the base of the abdomen in both males and females. These organs resemble the tymbals of male cicadas, but are not backed by large resonant tracheal air sacs and therefore cannot produce audible sounds. Energy is concentrated below 1 kHz, and is transmitted to the substrate through the legs or mouthpart stylets. (c) Green lacewings (Chrysoperla) jerk or oscillate their abdomens vertically at 30-120 Hz, shaking the substrate sympathetically39. Extremely sensitive mechanoreceptors in the tibiae of the legs are tuned sharply to the frequency range characteristic of the species, Long alternating heterosexual duets must precede copulation.
genotypes, each with separate and incompatible mate recognition systems. Support for certain aspects of this model exists in real insect groups, described below. Songs of Diptera Near-field songs have been well studied in the true flies (Diptera). Wing-flicking during courtship and mating occurs in both sexes of many drosophilids and tephritids (Fig. la), producing complex patterns of pulsed air over very short distances that are decoded by the antennaels. In typical small-bodied drosophilids, female singing and heterosexual dueting are commonplace. However, male songs are more elaborate and less variable (more stereotyped) than female signals, and songs differ most markedly between adjacent and sympatric specie+‘. Furthermore, vibrational and other acoustical signaling in the larger-bodied Hawaiian Drosophila have clearly evolved under traditional sexual selection, as components of rivalry or lekking displays performed principally by males*O.Thus, drosophilids do not conform well to the speciation model for silent singers suggested above. Such a finding is not unexpected: even small mainland drosophilids use wing-flicking partly as a visual or olfactory display, and their larger Hawaiian relatives produce clearly audible clicks and hums during courtship. The patterns of recent, rapid and repeated speciation seen in several groups of Drosophila are therefore more likely to have resulted from founder-flush events (rapid evolutionary divergence resulting from the relaxation of stabilizing selection after a founder event)zl, or from bouts of intense sexual selection mediated by other types of signals. 390
A few dipterans are known to use substrate-borne vibrational signals during courtship. Hawaiian Drosophila siloestris vibrate their abdomens to produce a low-frequency purr*“, while small chloropid flies of the genus Lipara produce trains of species-specific pulses through a similar mechanism. Receptive males and females of the latter typically establish duets before mating, although the female signal differs from that of the male in several respect+. Details of species isolation in Lipara are not known, but their reproductive behavior is consistent with speciation facilitated by silent songs. Songs of Auchenorrhyncha Many families of small Auchenorrhyncha (order Hemiptera), including planthoppers, leafhoppers, treehoppers and spittlebugs, use abdominal tymbals (Fig. lb) to produce substrate-borne vibrational signals during courtship and mating24.25.As in Diptera, the silent songs of auchenorrhynchs are often found in both sexes of a species and have been implicated in reproductive isolation and speciation. Typical of the tymbal singers is the brown planthopper (Nilczpatuatu/ugens)*G.This is a single morphological species, but in Australia it is subdivided into at least two acoustically distinct biological species, each associated with a different host plant. Courting partners engage in vibrational duets, and the songs of females are very similar to those of conspecific males. In broad areas of sympatry, male and female songs are the principal barriers to hybridization between these sibling species. Because song acts as a homogametic mechanism, a series of biological species have differentiated rapidly within Nilaparuata, possibly through incidental changes in song phenotype27. This example clearly provides support for a variant of the simple speciation model outlined earlier. It is plausible that noncontinuous, arbitrary changes in songs have cut off gene flow between populations that were initially associated with different host plants. A parallel situation is seen in another planthopper, Ribautodelphaxz8. Female singing and heterosexual dueting are important components of reproductive behavior, and the female song phenotype is highly species-specific. Males appear to be even more discriminating of song parameters than are females, reversing the normal logic of female choice. DeWinterz8 takes a broad view of sexual selection, in which species recognition is considered a sub set of female and/or male choice. Male choice in Ribautodelphux may then function principally in conspecific recognition, because it is advantageous for such decisions to be made early in courtship, which incidentally places a selective premium on precise, invariant female signalsZg. Sexual selection therefore acts on both sexes in different ways: males compete among themselves for access to females, while the reduced pool of females compete intrasexually so as to assure their rapid insemination by discriminating males. The concerted effect of these pressures on the song phenotype of both sexes could conceivably drive divergence and speciation very rapidly. Studies on other genera of planthoppers confirm this pervasive pattern of host-associated sibling species distinguished principally by their songs. Among many possible examples, one can cite Jauesella30 or ProkelisW, each of which contains several closely related dueting species that have been shown to be reproductively isolated from one another, at least in part, by their complex courtship songs. As in Nilaparuata, powerful, bidirectional mate choice could accelerate the reproductive divergence of populations that were initially associated with a series of host plants, resulting in rapid and repeated speciation events.
REVIEWS One final noteworthy example is drawn from the treehoppers (Membracidae). Like the planthopper taxa above, Enchenopa bino&ta is actually a complex of sympatric biological species, each affiliated with a different host plant species. The life history of each of the nine Enchenopu species is intimately associated with the phenology of its host; consequently, accidental host shifts apparently have a strong reproductive isolating effect, which could result in sympatric speciatior+““. Very recent work, however, has documented species-specific differences in the substrateborne songs of both males and females of Enchenopa, suggesting that song divergence has also influenced the process and pattern of evolution34. Although it was originally thought that host-associated factors had driven the species apart and now kept them reproductively isolated, vibrational songs are equally effective causes of assortative mating and speciation. The mere existence of such courtship signals had not been suspected when the evolution of this species complex was first investigated. Songs of Chrysopidae (Neuroptera) Communication through the substrate is part of the ground plan of Neuroptera and its plesiomorphic sister orders, Megaloptera and Raphidioptera. Members of all three groups produce low-frequency vibrational songs by vigorously jerking (but not drumming) the abdomen (Fig. lc). The best-studied taxon is Chrysoperla, in the diverse neuropteran family Chrysopidae (green lacewings). These insects generate volleys of abdominal vibration when sexually receptive”5. The song either consists of simple trains of one volley type, or is organized further into groups of volleys of one or more different types (Fig. 2). Songs are species-specific, and both males and females sing identically, alternating single volleys or groups of volleys in precise duets during courtship and mating. The carrier frequency typically sweeps up or down in pitch during each volley. Within the genus, sexual dimorphism is minimal, and there is little evidence of intrasexual competition for mates: both sexes are equally discriminating during courtship. Also, song differences between closely related species are not greater in sympatry than they are in allopatry36. As in the hoppers, morphological species are not necessarily congruent with biological species in Chrysoperiu. For example, Chrysoperla carnea is a single morphological species, with a holarctic distribution. Morphological homogeneity across this vast geographical range applies to the finest details of male and female genitalia, and gel electrophoresis has proven no more effective than morphology for delineating taxonomic units within the species37J8. However, in recent years, well-defined ‘song morphs’ of C. curnea have been discovered, each with consistently different courtship signals and extensive distributions. Unlike hoppers, these song morphs are not characterized by different ecological niches, and often coexist at a single site, even living together on the same individual tree or bush. At least five of them have been detected in North America and five more in Europe (Fig. 3). All efforts to find hybrids between the morphs in the field have failed, and the results of playback experiments indicate that songs have an extreme isolating effect in both sympatric and allopatric population+‘. Clearly, the song morphs of C. curnea are good biological species, comprising a swarm of sibling cryptic taxa”“. To conform to our model of speciation through song divergence, genetic control of song phenotype should be relatively simple. Fortunately, the song species of the C. curneu complex hybridize in the laboratory when given
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Time (seconds) Fig. 2. Oscillograph of the complete song of Chrysoperla]ohnsonF. showing typical song features used to distinguish the biological species of the C. camea species group. (a) volley duration; (b) volley repetition interval: (c), (d) and (e) frequency (pitch) of start, middle and end of volley, respectively: (f) duration of last songvolley; (g) number of volleys per SRU (shortest repeated unit of song); (h) SRU duration.
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Tme (seconds) Fig. 3. Oscillographs of the songs of nine biologtcal species of the C. camea species group, all drawn to the same scale (12 seconds). Species above the dotted line are from North America, while those below the ltne are from Europe17.
no choice, and F,, F, and backcross generations are viable and reasonably fertilel7.41.Available evidence is consistent with straightforward mendelian inheritance of song features at two or three different loci. Each species could be homozygous for a different set of alleles at each of those loci, which would produce the observed result: F, hybrids with intermediate and invariant song phenotypes [Fig. 4).
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REVIEWS A single gene mutation at a song-controlling locus would then alter singing behavior significantly, disrupting courtship between a mutant and a normal individual. A final consideration is the strength of assortative mating attributable to courtship songs. This has been tested in F, hybrids between two North American song species42, which discriminate as strongly against their parents’ songs as each of their parents do against the songs of non-conspecifics. It is therefore not so far-fetched to suggest that the gene for a new song in a population might quickly become fixed in a reproductively isolated subset of individuals possessing that song. Repeated occurrence in different regions would produce the swarms of closely related, sibling species that we see today.
General considerations I have suggested a new way of thinking about the process of speciation in insects characterized by inaudible, silent songs. The special acoustical and genetic characteristics of some of these insects can produce nearly instantaneous reproductive isolation of populations with new songs, unencumbered by adaptive responses to the environment, geographical isolation, reinforcement, repro ductive character displacement or genetic revolution. Unfortunately, in evolutionary biology, there is a disturbing tendency for each taxon to respond to its own set of rules, and it may be that Chrysoperla green lacewings are stolidly unique. Nonetheless, the data from other silently singing Diptera and Auchenorrhyncha summarized earlier
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are suggestive of alternative modes of speciation in insects
possessing vibrational or near-field courtship signals. A useful approach for the future is to assess the phylogenetic relationships among the silent singers in a given taxon, using independent criteria (e.g. molecular markers). By de termining the directions and pattern of speciation in each group, it will be possible to choose one model over another with more confidence. Acknowledgements For help in all aspects of the lacewing work described here, I thank Phillip A. Adams, Stephen Brooks, Peter Duelli, James B. Johnson, Raymond J. Pupedis and Marta M. Wells. Julie J. Henry and Marta M. Wells critically read and improved the manuscript. The research was supported in part by grants from the National Science Foundation (DEB-9220579) and the University of Connecticut Research Foundation (FRS440393).
References 1 Alcock, J. and Gwynne, D.T.(1991)in Reproductive Behaviour of insects (Bailey, W.J. and Ridsdill-Smith, J., eds), pp. 10-41, Chapman &Hall 2 Otte, D. (1992) J Orthoptera Res. 1,25-49 3 Lande, R. (1981) Proc. Nat/Acad. Sci. USA 78,3721-3725 4 West-Eberhard, M.J. (1984) in insect Communication (Lewis, T., ed.), pp. 283-324, Academic Press 5 Kirkpatrick, M. (1987) Annu. Rev Ecol. Syst. 18,43-70 6 Houde. A.E. (1993) Am. Nat. 141, 796-803 7 Ritchie, M.G.,Butlin, R.K. and Hewitt, GM. (1992) Biol. J Linn. Sec.
45,219-234 8 Butlin, R.K. (1987) Am. Nat. 130,461-464 9 Coyne, J.A. and Orr, H.A. (1989) Evolution 43,362-381 10 Walker, T.J. (1974)Am. Zoo/. 14,1137-1150 11 Forrest, T.G. (1991) Behav. Ecol. 2,327-338 12 Bennet-Clark, H.C. (1971) Nature 234,255-259 13 Stewart, K.W., Bottorff. R.L., Knight. A.W. and Moring, J.B. (1991) Ann. Entomol. Sot. Am. 84,201-206 14 Morris, G.K. (1980) Anim. Behov. 28,42-51 15 BennetClark,H.C.,Leroy,Y.and Tsacas, L (198O)Anim. Behov. 28,230-255 16 Paterson, H.E.H.(1986) in Species and Speciotion (Transuool Museum Monograph No. 4) (Vrba, E.S., ed.), pp. 21-29, Transvaal Museum 17 Henry, C.S. (1985) Euokttion 39,965-984 18 Sivinski, J., Burk, T. and Webb, J.C. (1984)Anim. Eehao. 32, 1011-1016 19 Ewing, A.W. (1970) Rev. Comport Anim. 4,3-8 20 Hoikkala, A., Hoy, R.R. and Kaneshiro, K.Y.(1989) Anim. Behav. 37. 927-934
123456789 Song phenotype
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-iiiilii Song phenotype Fig. 4. lnhentance
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301 x plorabunda
x downesi
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of song phenotypes In F,, F, and backcross progeny of Chrysoperla plorabunda x C. downesi, two North American sibling species of the C. carnea species complex. (a) Oscillographs of the songs of each species. (b) Oscillographs and phenotype frequencies of F, hybrids. (c) Phenotype frequencies of F, hybrids. (d) Phenotype frequencies of backcross progeny. Song phenotypes are classified into nine categories, based on a suite of features. The extremes, types 1 and 9, represent the songs of parental C. plorabunda and C. downesi, respectively17.
28 29 30 31 32 33 34 35 36 37
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Carson, H.L.and Templeton, A.R.(1984)Annu. Rev. Ecol. Syst. 1597-131 Hoy, R.R.,Hoikkala,A. and Kaneshiro, K.Y.(1988)Science 270,217-219 Kanmiya, K. (1990) J. Ethol. 8,105-l 19 Ossianilsson, F. (1949) Opusc. Entomot. Suppl. 10, 1-145 Hunt, R.E. (1993) Ann. Entomot. Sot. Am. 86,356-361 Claridge, M.F. (1990) Con. J Zool. 68, 1741-1746 Bailey, W.J. (1991) Acoustic Behoviour oflnsects: an Evolutionary Perspectiue, Chapman &Hall DeWinter, A.J. (1992) J Euo[. Biol. 5,249-265 Von Schilcher, F. and Dow, M. (1977)Zeitschr. Tierpsychoi.43,304-310 DeVrijer, P.W.F. (1986) Neth. J. Zool. 36, 168-179 Heady, S.E. and Denno, R.F. (1991) J. Insect Behav. 4,367-390 Wood, T.K. and Keese, M. (1990) Evolution 44,619-628 Wood, T.K. (1993) Annu. Rev. Entomot. 38,409-435 Hunt, R.E.J NY. Entomot. Sot. (in press) Henry, C.S. (1980)Ann. Entomot. Sot. Am. 73,617-621 Henry, C.S. and Wells, M.M.(1990)Ann. Entomot. Sot. Am. 83,317-325 Bullini, L.. Principi, M.M.and Cianchi, R. (1984) in h-ogress in World’s Neuropterotogy (Gepp, J., Asp&k, H. and Holzel, H., eds), pp. 57-59, Thalerhof Wells, M.M.Ann. Entomol. Sot. Am. (in press) Wells, M.M. and Henry, C.S. (1992) Evolurion 46,31-42 Henry, C.S., Wells, M.M.and Pupedis, R.J. (1993) Ann. Entomot. Sot. Am. 86, l-13
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Wells, M.M. (1993) Can. J Zoot. 71, 233-237 Wells, M.M. and Henry, C.S. J fnsect Behau. (in press)