Obligatory Duetting Behaviour in the Chrysoperla Carneagroup of Cryptic

Home , Chrysoperla carnea, Gryllus fultoni

Biol. Rev. (2013), 88, pp. 787–808. 787 doi: 10.1111/brv.12027 Obligatory duetting behaviour in the Chrysoperla carnea-group of cryptic species (Neuroptera: Chrysopidae): its role in shaping evolutionary history

Charles S. Henry1,∗, Stephen J. Brooks2, Peter Duelli3, James B. Johnson4, Marta M. Wells5 and Atsushi Mochizuki6 1 Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, Connecticut CT 06269, U.S.A. 2 Department of Entomology, The Natural History Museum, London, SW7 5BD, U.K. 3Swiss Federal Research Institute WSL, Birmensdorf, CH-8903, Switzerland 4Division of Entomology, University of Idaho, Moscow, Idaho, ID 83844, U.S.A. 5Department of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut, CT 06520, U.S.A. 6National Institute for Agro-Environmental Sciences, Tsukuba City, Ibaraki, 305 8604, Japan

ABSTRACT

An unusual system of communication has evolved in green lacewings of the Chrysoperla carnea-group, triggering rapid proliferation of numerous cryptic species across all of the Northern Hemisphere and large portions of Africa. The system is based on sexually monomorphic, substrate-borne vibrational signals, produced by abdominal oscillation. These low-frequency signals are exchanged between courting individuals in a precise duetting format. The song of each of the more than 20 described species exhibits a unique acoustical phenotype that reproductively isolates the taxon from all other species with which it might come into contact. Here, we review what is known about duetting behaviour in the carnea-group, emphasizing the dominant role that duetting has played in the evolution, ecology, and speciation of the complex. Included are descriptions and discussions of song diversity and its impact on reproductive isolation among species, the genetic basis of interspeciﬁc song differences, partitioning of acoustic space among sympatric species, parallel song evolution in allopatric species pairs, and modes of speciation within the complex. We also emphasize the importance of correctly identifying by song all species of the carnea-group that are to be used either in experimental studies or programs of biological control, while acknowledging the continuing relevance of morphology to carnea-group systematics.

Key words: songs, speciation, insects, taxonomy, convergent evolution, parallel evolution, sibling species, sexual monomorphism.

CONTENTS I. Introduction ...... 788 II. The Chrysoperla carnea (Stephens) species-group ...... 789 III. Sexual signalling and the duetting songs of thecarnea-group ...... 789 (1) Song production in insects ...... 789 (2) Communication between the sexes ...... 790 (3) Sexually monomorphic duetting songs ...... 790 (4) Multimodal sexual signalling ...... 791

* Author for correspondence (Tel: ++86 04 864450; E-mail: [email protected]).

IV. Diversity of duetting song phenotypes in the carnea-group ...... 791 V. song variation within species ...... 793 (1) Geographic variation ...... 793 (2) Phenotypic plasticity ...... 795 VI. reproductive isolation ...... 796 (1) Premating, prezygotic isolation ...... 796 (2) Postmating, postzygotic isolation ...... 796 VII. Acoustic space and song evolution ...... 797 (1) Parallel evolution ...... 798 (2) Movement between continents ...... 798 VIII. Duetting and speciation ...... 799 (1) Three critical properties of the clade ...... 799 (a) Duets enforce reproductive isolation ...... 799 (b) The genetic basis of song differences is simple ...... 799 (c) Cryptic phenotypes have cryptic genotypes ...... 800 (2) Mode of speciation ...... 800 (3) Ecology and speciation ...... 800 (4) Does reinforcement complete speciation? ...... 801 IX. The importance of morphology ...... 802 X. Denying song-delineated species: studies from the literature ...... 803 XI. Cryptic species, systematics, and biological control ...... 804 XII. Future directions ...... 804 XIII. Conclusions ...... 804 XIV. Acknowledgements ...... 805 XV. References ...... 805

of cryptic species, each with a species-specific duetting ‘When they came near each other, both the males and the song (Henry, 1983a). This complex is the focus of our females began to jerk the abdomen upward and downward, review. First, we present the classification of the carnea- an act which is generally seen before copulation.’ group. Because of the cryptic nature of these species, the fast-evolving duetting signals are the most reliable (Smith, 1922, pp. 1331–1332) taxonomic characters in the clade; therefore, we summarize sexual signalling and song diversity, highlighting the features I. INTRODUCTION that make the song of each species unique. We describe intraspecific variation in duetting behaviour, parsing the Written 90 years ago, these words record the observations relative contributions of genetic versus environmental effects made in 1916 by Dr Roger C. Smith of Cornell University and reviewing the results of single-species experiments on concerning behaviour he had seen in a common North sex recognition and intrasexual competition. The impact of American insect species, Chrysopa oculata Say. In publishing duetting songs on premating reproductive isolation between this paper, Smith became the first scientist to describe otherwise interfertile cryptic species is then described and courtship and mating in a green lacewing (Order Neuroptera, its implications discussed, especially within the context of Family Chrysopidae). More significantly, his account is the relatively weak postmating, postzygotic isolation across the first to identify the essential elements of a remarkable system carnea-group. Because duetting songs occupy distinct regions of duetting behaviour in lacewings, one that is based on of acoustical space, we explore the physical and biotic nature substrate-borne signals or ‘songs’ produced by the vibrating of this space, the importance of species-specific regions abdomen of sexually active individuals. of space, and the impact of available acoustic space on It would be another 40 years before Smith’s observations parallel (or convergent) song evolution in species that will not were confirmed and extended to other species and genera encounter each other because of their allopatric distributions. within the Chrysopidae (Toschi, 1965; Riek, 1967; Ickert, One likely consequence of ongoing song divergence in the 1968; Sheldon & MacLeod, 1974; Brooks, 1987). Even so, carnea-group is recent, rapid speciation, so we examine some and despite a growing body of literature (see references in newer ideas about song-mediated cladogenesis in lacewings. Henry & Wells, 2010), the nature of duetting behaviour and Throughout the review, we emphasize the necessity of using its impact on the evolution, speciation, and classification an accurate taxonomy of carnea-group lacewings based on of lacewings are not familiar to many biologists. One song-delineated species, in which morphology and ecology particular clade of lacewings, the carnea-group of the are accorded secondary rather than primary taxonomic genus Chrysoperla Steinmann, has radiated rapidly and importance. We show how the identification of cryptic species recently into an especially large and important complex can sometimes be achieved even using dead specimens,

Biological Reviews 88 (2013) 787–808 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society Duetting and evolution in lacewings 789 if morphological character states are analyzed as correlates of evidence in the carnea-group, which he attributed to its specific song phenotypes. The pitfalls of incorrect taxonomy retention of ‘many plesiomorphic chrysopine characters’. are highlighted using case studies from the literature, in which Fortunately, the carnea-group is specialized in at least one careful observation and experimentation have unfortunately way: its complex, substrate-borne, low-frequency duetting produced ambiguous and confusing results rather than songs. In this respect its morphologically cryptic species are useful insights. Duetting song taxonomy must also serve rich in derived characters, and during the last 30 years as the foundation for effective and responsible programs these song traits have been used with increasing success to of integrated pest management (IPM), not only to achieve clarify the boundaries between existing taxa and to erect maximum commercial success but also to avoid introducing new species when necessary (Henry et al., 2012). Today, the non-native cryptic species that could displace and eliminate carnea-group alone comprises 21 valid described species. In native ones. Finally, we identify critical research areas where addition, several new cryptic taxa have been identified but additional studies are badly needed. not yet described. Poor sampling of the carnea-group across In short, green lacewings of the Chrysoperla carnea-group much of Asia, northern and central Africa, and western have proven to be an ideal system within which to study North America makes it very likely that many new species speciation. They are a natural experiment in progress, remain to be discovered. where variables such as morphology and ecology are held constant and only the fast-evolving mating signals remain as a significant source of variation. Furthermore, the ability that we have to manipulate these signals makes it possible to III. SEXUAL SIGNALLING AND THE DUETTING study their precise role in species origins. SONGS OF THECARNEA-GROUP

(1) Song production in insects II. THE CHRYSOPERLA CARNEA (STEPHENS) SPECIES-GROUP Singing is widespread in insects. The sclerotized cuticle pre-adapts them for audible sound production via scraping Chrysoperla is one of approximately 75 described genera in actions (Henry, 2006), and many familiar insect singers the neuropteran family Chrysopidae (Brooks & Barnard, like crickets, grasshoppers, and katydids (Orthoptera) rub 1990). Members of the genus are unusual among green wings against wings or legs against wings or abdomen to lacewings in that they overwinter as adults. They are create airborne sound signals (Otte, 1993). Other notable predatoryonlyaslarvae(Sem´ eria,´ 1977), and the adults singers like the cicadas (Hemiptera) sing loudly by rapid and are honeydew feeders (but see Tauber & Tauber, 1973). repeated buckling of a stiff drum-like cuticular structure, the In his monograph, Brooks (1994) recognized 36 species of tymbal (Sueur & Aubin, 2003). However, communication Chrysoperla, organized into four species-groups based largely via audible airborne sound is not practical in smaller insects, on male genitalic characters. With respect to morphology, because at small size it becomes energetically too costly to the least derived of these four is the carnea-group, named for sing aloud at frequencies below 10–15 kHz (Virant-Doberlet its best-known constituent species, C. carnea (Stephens). As & Cokl, 2004). Also, not all insects are hard-bodied – and this Smith (1932, p. 592) presciently stated years ago, ‘This group is particularly true of smaller insects. Nonetheless, small or [comprising species similar to C. carnea] contains several very soft-bodied species can sing, but to do so efficiently they must perplexing, variable species .... No satisfactory morphological propagate lower-frequency signals through a solid substrate characters which can be used as species criteria have yet been rather than through air (Cocroft & De Luca, 2006). For discovered.’ The truth of this statement has been confirmed example, among the Hemiptera, many leaf hoppers and plant repeatedly over the years, as taxonomists divided ‘C. carnea’ hoppers (Auchenorrhyncha: Cicadellidae and Fulgoroidea) (s. lat.) into many questionable species and varieties. Indeed, ‘buzz’ plant surfaces using scaled-down versions of the tymbal at least 15 synonyms of the species were listed by Aspock,¨ found in their larger cicada relatives (Virant-Doberlet & Holzel¨ & Aspock¨ (2001) in a recent catalogue. Cokl, 2004), while most tree hoppers (Auchenorrhyncha: In reaction to what he considered to be the unjustified Membracidae) and stink bugs (Heteroptera: Pentatomidae) proliferation of names for carnea-like species, Tjeder (1960) apparently press their vibrating abdomens against the placed most of them back into C. carnea s. str., rendering the substrate instead (Cocroft, Rodriguez & Hunt, 2010; Gogala, latter a single monolithic species with a Holarctic distribution. 2006). However, building on Tjeder’s innovative interpretation Green lacewings are both small and soft-bodied, so their of the male genitalia (Tjeder, 1954, 1966) and adding singing options are doubly limited. Consequently, they have characters of his own, Brooks (1994) was finally able to evolved to oscillate their abdomens at low frequencies and detect reliable taxonomic structure within the carnea-group. ‘shake’ the substrate, thereby producing bending waves in the His results supported re-dividing Holarctic C. carnea s. str. grasses, leaves, and plant stems on which they perch (Busnel, into a number of previously recognized but freshly delineated Pasquinelly & Dumortier, 1955; Cocroft & Rodríguez, 2005; species. Nonetheless, like Smith (1932), Brooks (1994, p. 142) Michelsen et al., 1982). These tremulation signals are easily acknowledged the often inconclusive nature of morphological detected and recorded using a transducer that lightly touches

Biological Reviews 88 (2013) 787–808 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society 790 Charles S. Henry and others the vibrating substrate, such as a piezoelectric accelerometer 1980; Henry, 1994; but see Laumann et al., 2011 for a or phonograph cartridge (Gogala, 2006). Alternatively, one counterexample). However, even when both sexes participate can use a non-contact device that monitors the reflection in an obligatory vibrational or acoustical duet, the exchange is of a beam of light directed at the substrate, such as a usually asymmetrical – males typically produce longer, more laser vibrometer (Mazzoni et al., 2010) or infrared optical complex, and/or more intense signals than females (e.g. de microphone (Fig. 1, see also Henry & Wells, 2006). Groot et al., 2012; Sandberg & Stewart, 2005; Forrest, Lajoie & Cusick, 2006; Kanmiya, 2006; Percy, Taylor & Kennedy, (2) Communication between the sexes 2006; Rodriguez & Cocroft, 2006; Tishechkin, 2006; Orci, 2007; Mazzoni et al., 2009, 2010). Insect species sing to locate, attract, and court sexual partners, as well as to secure mating opportunities through success (3) Sexually monomorphic duetting songs in intrasexual competition (an excellent review of animal communication can be found in Bradbury & Vehrencamp, Remarkably, lacewings of the carnea-group are among the 2011). Usually, conspicuous singing in adults is restricted to very few singing insects characterized simultaneously by true the male sex of a species, because high variance in male duetting behaviour and sexually monomorphic, complex reproductive success leads indirectly to riskier behaviour songs (Noh & Henry, 2010). In each species, males and (Bailey, 2006). In those cases, females either orient silently females produce very similar vibrational songs that alternate to male sounds or respond irregularly using simpler signals between the courting individuals in a polite, non-overlapping of low amplitude, thereby avoiding both the energetic costs manner (Fig. 2). The portion of the song that serves of sound production and the risk of detection by acoustically as the unit of exchange between partners during a duet orienting predators or parasites (Zuk & Kolluru, 1998; is called the ‘shortest repeated unit’ (SRU). The SRU Tinghitella & Zuk, 2009). has a species-specific temporal and frequency structure. More rarely, a singing species will have true duetting It is made up of one to many discrete ‘bursts’ or volleys behaviour, defined as an interaction between the adult of abdominal vibration, each having a certain duration, male and female that is ‘ ... marked by the predictable repetition rate, and carrier frequency. Elaborations of this and stereotyped temporal association between initiating call template, found in many species, include the presence of and the reply’ (Bailey, 2003, p. 157). Such heterosexual several different volley types within the SRU (e.g. C. downesi duetting has evolved more often and to a greater degree in Fig. 2), or the expression of directional changes to basic of complexity in insects that communicate vibrationally, temporal and frequency features during the course of the presumably because a female’s song is less likely to be SRU (e.g. nearly all the species shown in Fig. 3). Thus, noticed by predators and parasites when it is confined patterns of duetting range from the rapid-fire exchange to the local substrate (Bennet-Clark, Leroy & Tsacas, of short, single-volley SRUs in C. plorabunda and ‘CcX- Asia’, to the drawn-out exchange of very long, multi-volley SRUs in C. downesi, C. carnea, C. mediterranea, C. pallida,and C. calocedrii (Figs 2 and 3). (Note that author citations for all Chrysoperla species names used in this paper are given in Electronic Table 1.) shaker Optical Consistent with monomorphism, males and females of (beneath microphone output arena) Power amp carnea-group taxa are equally likely to sing spontaneously and therefore to initiate a duet with another individual. input IR reflective beam membrane However, unless the partner is a member of the same species and sings the same song as the initiator, a duet will not be established. Failure to duet is largely because of irreconcilable differences in the length of the SRUs and the timing of volleys within them (i.e. duration, period, and temporal arrangement CED digitizer/ Arena Low-pass filter Computer of distinct volley types; see Section IV). However, volley (cardboard cup) output carrier frequency can also be important, particularly if it is input modulated up or down within the volley or during the course of the SRU. Thus in C. plorabunda, both the volley period and the presence of frequency modulation within the volley have Fig. 1. Diagrammatic sketch of the apparatus used to record been shown to be critically important for species recognition and play back substrate-borne vibrational duetting signals (Wells & Henry, 1992a). produced by green lacewings. The optical microphone detects One expected consequence of song monomorphism is an infrared (IR) light beam reflected from the plastic membrane incorrect sex recognition during acoustic encounters between covering the experimental arena. The electronic shaker, used for individuals. Thus, homosexual duets are commonly observed inducing vibrational signals in the lacewing’s substrate during in most if not all species of the carnea-group (first described playback experiments, is actually positioned beneath the arena. in Henry, 1979). Although such duets can be lengthy in CED = Cambridge Electronic Design, Ltd; amp = amplifier. either sex, those between females are usually the longest,

C. plorabunda C. downesi, detail volley period ( ) 120 Hz 100 80 60 40 long volley short volleys 20

SRU volley

C. downesi

0 1 23 4 5 6 7 8 901 11 12 13 14 15 16 17 s Time (s) Fig. 2. Oscillograms of heterosexual duets in two species of the carnea-group, drawn to the same time scale (bottom of figure). The upper trace, of C. plorabunda, shows a duetting interaction in a species that has a single-volley shortest repeated unit (SRU, see text). The lower trace, of C. downesi, shows a duetting interaction in a species having a multi-volley SRU consisting of two different types of volleys. A magnified one-second detail of the two types of volley in the C. downesi song is shown in the upper right corner of the figure; it includes both an oscillogram view (lower) and a spectrogram view (upper) of the signal. occasionally exceeding five minutes in C. plorabunda (Henry IV. DIVERSITY OF DUETTING SONG & Wells, 2009) and C. carnea (C. S. Henry, unpublished PHENOTYPES IN THE CARNEA-GROUP data). Interestingly, males of C. plorabunda often break out of homosexual duets by mutually and progressively decreasing As few as 5 to as many as 19 distinct, measurable features volley period to the point of duet failure, thus indirectly distinguish the duetting songs of different species of the allowing an individual to determine the sex of its partner carnea-group from one another (Henry et al., 2012). In (Henry & Wells, 2009). Subtle deviations from complete a species having a simple, single-volley SRU, the song sexual monomorphism, exemplified by such male-limited phenotype may be described fully by volley duration, volley ‘fast duets’, deserve investigation in other species of the period, and the carrier frequency measured at the beginning, carnea-group as well. middle, and end of the volley (Figs 2 and 3). However, in a species characterized by more than one volley type and (4) Multimodal sexual signalling by long, multi-volley SRUs, those five measurements must be applied independently to each volley type, as well as to There is no evidence explicitly supporting other modalities volleys at the beginning, middle, and end of the SRU as of intraspecific communication that supplement or replace a whole. In addition, a multi-volley SRU will have its own duetting behaviour in any members of the carnea-group. duration and period, independent of volley traits. Nevertheless, pheromones and cuticular hydrocarbons are Although they vary in complexity, even the simplest known to be part of courtship and mating in other duetting songs must be viewed as multidimensional suites Chrysopidae (e.g. Glenochrysa spp. Esben-Petersen, Meleoma of phenotypic traits. Thus differences among species are best emuncta (Fitch), and Chrysopa spp. Leach; see Tauber, analyzed using multivariate statistical methods, especially 1969; Wattebled & Canard, 1981; Duelli, 2004; Zhang techniques that classify and parse the variance of multiple et al., 2006a,b). Like these other genera, Chrysoperla also dimensions into two or three principal components or possesses a large prothoracic gland that secretes a diverse discriminant factors (e.g. see Figs 8 and 9 in Henry & Wells, array of semiochemicals (Aldrich et al., 2009), suggesting 2010). In that way, we can more clearly visualize the relative that multimodal courtship and mating cannot be ruled positions of the songs in a kind of ‘acoustical space’ (Duellman out in this genus. However, vibrational duetting, which & Pyles, 1983), akin to the more familiar concept of niche continues uninterrupted from the earliest stages of courtship space in ecology (Bambach, 1983; Hutchinson, 1957). In the until copulation, seems to provide most if not all of absence of other forms of reproductive isolation, two species the information necessary for mating decisions in the in the carnea-group should not be able to occupy the same carnea-group. region of acoustical space simultaneously (see Section VII).

Oscillograms Spectrograms

140 100 C. plorabunda 60 (Fitch) 20 140 100

aciremAhtroN C. adamsi 60 Henry et al. 20 140 100 C. johnsoni 60 Henry et al. 20 140 100 C. “downesi-2” 60 20

140 100 C. downesi /“mohave” 60 (Smith)/(Banks) 20 140 100 C. calocedrii 60 20 Hz Henry et al.

140 100 “CcX-Asia” 60 20

140 100 C. “adamsi-Kyrg.” 60 20

140 100 C. z. sillemi 60 (Esben-Petersen) 20 140 100 C. z. zastrowi 60 (Esben-Petersen) 20 140 100 C. agilis 60 20 acirfA& aisaruE acirfA& Henry et al. 140 100 C. lucasina 60 (Lacroix) 20

140 100 C. “nipponensis-B” 60 20

140 100 C. nipponensis 60 (Okamoto) 20 140 100 C. “downesi-Kyrg.” 60 20 140 100 C. pallida 60 Henry et al. 20 140 100 C. “carnea-Kyrg.” 60 20

140 100 C. carnea 60 (Stephens) 20 140 100 C. mediterranea 60 20 Hz (Hölzel)

024681012s 024s Fig. 3. Oscillograms and spectrograms of species-speciﬁc duetting songs recorded from 19 species and subspecies of the carnea-group. The taxa are grouped by continent, and within continents by song complexity. The arrows mark the points at which the partner in the duet inserts its SRU (= song); single-volley species reply after each of the partner’s volleys, while multi-volley species reply when the partner’s shortest repeated unit (SRU) is ﬁnished.

Multivariate methods are powerful discriminators of song short, single-volley SRUs. Placed in the following order phenotypes, but traditional oscillograms (signal amplitude below the top are longer single-volley SRUs, multiple-long- versus time) and spectrograms (signal frequency versus time) volley SRUs , multi-volley SRUs made up of more than one are enough to conﬁrm high levels of diversity and disparity volley type, and multiple-short-volley SRUs. Arrows show among the songs of these cryptic species. In Fig. 3, graphical where the partner enters the duet. Among all the known views of the songs have been organized ﬁrst by continent species, C. plorabunda from North America and ‘CcX-Asia’ and then by song structure. Thus within each continent we from Caucasus and Iran have the simplest, shortest, single- show the simplest songs at the top – that is those having volley SRU songs, while the most complex songs are found

Table 1. Recognized species and subspecies of the carnea-group, with citations to literature where their duetting songs are described

C. plorabunda (Fitch, 1855) Henry (1985b), Henry & Wells (1990), Henry et al. (1993) and Wells & Henry (1992b) C. adamsi Henry et al. (1993) Henry (1991), Henry et al. (1993) and Wells & Henry (1992b) C. johnsoni Henry et al. (1993) Henry (1993a), Henry et al. (1993) and Wells & Henry (1992b) C. johnsoni ‘mohave ecotype’ Henry (1993b) C. ‘downesi-2’ (= ‘western downesi’) Henry (1985a,b) C. downesi (Smith, 1932) Henry (1980) C. downesi ‘mohave ecotype’ (Banks, 1938) Henry (1993b) C. calocedrii Henry et al. (2012) Henry et al. (2012) ‘CcX-Asia’ (present review) C. ‘adamsi-Kyrgyzstan’ Henry et al. (1999b) C. zastrowi sillemi (Esben-Petersen, 1935) Henry et al. (2006) and Henry et al. (2010) C. zastrowi zastrowi (Esben-Petersen, 1928) Henry et al. (2006) C. agilis Henry et al. (2003) Henry et al. (2003) and Henry et al. (2011) C. lucasina (Lacroix, 1912) Henry et al. (1996) C. ‘nipponensis B’ Henry et al. (2009) C. nipponensis (Okamoto, 1914) Henry et al. (2009) C. ‘downesi-Kyrgyzstan’ Henry & Wells (2004) and Wells & Henry (1998) C. pallida Henry et al. (2002) Henry et al. (2002a) C. ‘carnea-Kyrgyzstan’ (= ‘motorboat KR’) Wells & Henry (1998) C. carnea (Stephens, 1936) Henry et al. (2002a) C. mediterranea (Holzel,¨ 1972) Henry et al. (1999a)

in North American C. downesi and in Asian C. nipponesis, Table 1 cites the primary literature where complete song C. ‘nipponensis-B’, and C. ‘downesi-Kyrgyzstan’. descriptions can be found for each recognized species of The spectral (frequency) features of the songs are shown the carnea-group. Included in the table are several unnamed to the right of the oscillograms in Fig. 3. Other than being species that definitely have unique duetting signals but cannot limited to frequencies between 25 and 140 Hz, there are very be described properly until additional material becomes few constrained elements. Signals can be narrowly tuned (C. available. z. zastrowi), broadly tuned (C. calocedrii and C. pallida), or rich in harmonics (C. johnsoni and C. agilis); frequencies can rise or fall rapidly or slowly within a volley or across a multi- V. SONG VARIATION WITHIN SPECIES volley SRU (C. plorabunda, C. adamsi, C. lucasina, C. carnea, and C. pallida); or the spectral signature can quickly change Phenotypic variation has both genetic and environmental once or several times within the SRU (C. nipponensis and causes. At a given geographic site, the local population might C. ‘nipponensis-B’). It is also clear from Fig. 3 that those include an array of genotypes, expressed as quantifiable species whose songs look similar as oscillograms are usually variation among individuals in the feature of interest. Within easy to identify from their distinct spectrograms (e.g. C. carnea each individual, phenotypic variation can also arise from and C. mediterranea). an immediate or delayed plastic response to changes in There is little evidence that adaptation to environmental environmental conditions. In lacewings of the carnea-group, carnea properties has shaped signal structure in the -group, in each source of variation contributes to intraspecific duetting contrast to what has been found in many other animals variation, and each source has received attention in at least (Boughman, 2002; Endler & Basolo, 1998; Henry & a few taxa. Lucas, 2008; Sullivan-Beckers & Cocroft, 2010). Despite an intriguing and suggestive pattern of phenotypic resemblance among the duetting songs of lacewing species associated (1) Geographic variation with conifers (e.g. C. downesi, C. calocedrii, C. ‘downesi- Each song-delineated species in the carnea-group typically has Kyrgyzstan’, and C. mediterranea), no differences in either a very broad geographic distribution within a continent or efficiency of transmission nor signal preference could be global hemisphere, often spanning many thousands of linear detected when a conifer-associated species was compared to a kilometers (Fig. 4). For example, the dark-green form of grass-associated species (C. downesi versus C. plorabunda;Henry C. downesi in North America ranges from Connecticut in the & Wells, 2004). Thus, the signal properties of duetting songs east, northward through Canada to central Alaska (Henry, appear to have diverged randomly rather than adaptively 1980; Henry et al., 2011), then southward along conifer- with respect to the signal-transmission environment, to covered mountains into southern California. Another species create the diverse array of forms that we see today in the with an enormous distribution is the common Eurasian complex. species C. agilis, which ranges from the Azores in the west

adnubarolp .C adnubarolp .C ismada

aciremAhtroN

inosnhoj .C inosnhoj .C isenwod

iwortsaz .C iwortsaz .C siliga

? ? ? ?

anisacul .C anisacul .C aenrac

? ? ? ? acirfA& aisaruE acirfA& ? ? ?

llap .C llap adi .C aenarretidem

? ?

C. nipponensis Miscellaneous

? ?

= “CcX-Asia” = 3 Kyrgyzstan species = C. calocedrii =C. “nipponensis-B”

Fig. 4. Known geographic ranges (shaded) of 15 species of carnea-group green lacewings. Arrows point to the apparently isolated population of C. agilis in central Alaska and to the offshore populations of C. zastrowi in the Atlantic ocean (Cape Verde, Ascension, and St. Helena islands). across all of central/southern Europe and eastward to Iran; 2003, 2006, 2009; Henry & Wells, 2010). The existence there is also an established population of the species in of such relatively invariant duetting phenotypes within central Alaska, of uncertain origin (Henry et al., 2003, 2011). species suggests that significant levels of gene flow must C. zastrowi (including subspecies zastrowi and sillemi)isalso be maintaining the character states across great distances, remarkably widespread, occurring abundantly across the presumably due to the exceptional dispersal abilities of entire Indian subcontinent, the Middle East, and northern individual lacewings (for more on this, see Duelli, 1980a,b; and eastern Africa to its southernmost tip (Henry et al., 2006, Chapman et al., 2006). Also, strong stabilizing selection is 2010). probably acting to constrain further the song phenotype Despite such huge species ranges, lacewing duetting songs within each species, as indicated by consistent rejection generally show low levels of intraspecific variation, averaging of non-conspecific songs in playback experiments (Wells & 12–20% for temporal features and 2–12% for frequency Henry, 1992b, and discussed in Section VI). features (coefficients of variation measured in 12 species Only two species are known in which measurable by Wells & Henry, 1992b;Henryet al., 1999a, 2002a, clinal variation in song phenotype exists, instead of song

6.5 Chrysoperla plorabunda Chrysoperla adamsi (1) 6.0 1.00

5.5 R2=0.57, N=100 0.80 5.0 2.19 s 1.23 s

4.5 3.34 s northern 0.60 4.0 0.40 Volley period (s) 3.5 southern (1) C. plorabunda 3.0 0.20 C. adamsi F1 hybrid

2.5 Proportion of responses to stimulus 32 34 36 38 40 42 44 46 48 0.00 Degrees North latitude 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Volley period of stimulus (s) Fig. 5. Effect of latitude on volley duration and volley period in C. johnsoni from the western U.S.A. Each symbol represents Fig. 6. Plot of the mean proportion of duetting responses of the mean of the means of all individuals at a given latitude. The C. plorabunda (21 individuals), C. adamsi (26 individuals), and error bar shows the standard error of the mean. Oscillograms, plorabunda-adamsi F1 hybrids (18 individuals) given to stimulus drawn to the same time scale, compare the duetting songs of signals administered as tests of song preference over a normal typical northern versus southern representatives of C. johnsoni. range of volley periods for each species/hybrid. Vertical dotted Data are from Henry (1993a). lines mark the mean volley period for each of the three groups. Oscillograms, drawn to the same time scale, compare typical duetting songs of C. plorabunda and C. adamsi. Note that the homogeneity across the species range. One of these, C. johnsoni preference functions for C. plorabunda and C. adamsi do not (Fig. 5), produces SRUs comprising longer, slower-paced, overlap. Data are from Henry & Wells (2010). and (usually) more volleys as one travels south from northern Idaho to southwestern Arizona, U.S.A. (Henry, 1993a). The other, C. zastrowi, produces single-volley SRUs that are functions do not overlap (Fig. 6), indicating that the two more than 1.6 times as long in South Africa as they are in species will show strong reproductive isolation in nature. the Middle East and India (Henry et al., 2006, 2010). In this However, laboratory F1 hybrids between C. plorabunda and second species, the differences are sufficient to have prompted C. adamsi, which are characterized by intermediate songs recognition of two subspecies, C. z. sillemi in the Northern (mean volley period = 2.19 s), made acceptable duetting Hemisphere and C. z. zastrowi in the Southern Hemisphere partners for both parental species. Lack of strong behavioural (Figs 3 and 4), which also differ in some morphological isolation between parents and their hybrid progeny implies characters. that C. plorabunda and C. adamsi are sufficiently similar in song phenotype to prevent utilization of the signal space between them by a third species (see Section VII). Thus, plasticity is (2) Phenotypic plasticity high enough to facilitate tempo matching during conspecific In most carnea-group species, intraspecific variation in song duets, but low enough to keep the two species from mating features is not significantly greater than variation expressed with each other. as song plasticity within each individual (e.g. C. carnea It has been suggested that the properties of duetting and C. pallida,Henryet al., 2002a). However, at least songs might vary plastically in response to seasonal or other some plasticity of volley period and duration is particularly environmental factors (Tauber & Tauber, 1992; Tauber important in species having single-volley SRUs, because in et al., 2000). If that were true, song phenotype would those taxa it allows each individual to adjust its own singing be an unreliable premating barrier and would therefore tempo to that of its partner. As a result, a stable, persistent be of questionable taxonomic value. Ambient temperature, duet can be quickly established between the participants. for example, is known to be strongly correlated with song The dynamics of individual song matching during duets properties in poikilothermic arthropods, and lacewings are has been examined in detail in the sympatric North American no exception: volley duration, period, and carrier frequency species C. plorabunda and C. adamsi (Henry & Wells, 2006, scale linearly with temperature in all species (e.g. C. plorabunda 2010). These two species have similar single-volley songs that and C. adamsi, Fig. 7: data from Henry, 1991; Wells & Henry, differ in tempo, i.e. volley (and SRU) period averages 1.23 s 1992b). However, such temperature-related variation has no in C. plorabunda, but 3.34 s in C. adamsi (vertical dotted lines in effect on conspecific duetting ability in any species of the Fig. 6). Because of plasticity, individuals of each species can carnea-group, because temperature alters the song phenotype be manipulated to duet correctly with playback signals that of each participant in the duet by the same amount (Henry, are well outside their normal range of tempos; in fact, there is 1983b). These and other observations also support the notion a narrow range of intermediate volley periods to which both that potential mates recognize each other largely because species will respond. Although such overlap in duetting ability their songs match, and not because they recognize specific implies the possibility of hybridization, individual preference details of song structure per se.

6.0 species on the same day from a single shrub or tree branch r 2=0.64, C. plorabunda (Wells & Henry, 1998). Thus, there is ample opportunity 5.0 N=56 C. adamsi for interspecific contact between such intimately sympatric populations. 4.0 Nevertheless, hybrids have not been found in nature. This is not because hybrid crosses are impossible, infertile, 3.0 r 2=0.77, N=34 or unproductive – in fact any two cryptic species, even 2.0 those native to different continents, will eventually hybridize Volley period (s) when confined with no conspecific option in a small arena, 1.0 producing numerous healthy progeny that are themselves capable of having viable offspring (Henry, 1985a, see Section 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 VI.2). Powerful premating reproductive barriers must be Temperature (°C) in place that prevent individuals of different species from mating. In nature, the duetting songs provide that function: Fig. 7. Effect of temperature on volley period in C. plorabunda if two individuals are unable to establish a duet of alternating and C. adamsi from the western U.S.A. Each symbol represents the mean value of the volley period of an individual at a given responses (SRUs), neither will continue singing, and each temperature. Data are from Henry (1991) and Wells & Henry will typically fly away. (1992b). The strength of premating reproductive isolation has been assessed largely through playback experiments that test the responsiveness of individuals of each of two species Potentially more serious is the impact of temperature on to recordings of one another’s songs (SRUs). Each insect developmental processes. Walker (1998, 2000) has shown is presented with its own (conspecific) song type and the that calling song pulse rate is significantly different (at the heterospecific song type in a paired design (Wells & Henry, same temperature) in spring versus autumn broods of the 1992b, 1994). The mean proportion of correctly timed partially sympatric cricket species Gryllus rubens (Scudder) and duetting responses given by each individual to conspecific Gryllus texensis (Cade & Otte). This remarkable finding implies versus heterospecific song types is then calculated across that rearing temperature could have a significant effect on several replicates, and those means are compared using the accuracy of species recognition among closely related, appropriate statistical methods. sympatric species. However, carnea-group lacewings are more Thus far, such tests have been administered to all pairwise similar to Hawaiian Laupala crickets (Fergus, Decarvalho combinations of C. agilis, C. lucasina, C. pallida, C. carnea,and & Shaw, 2011) in showing no evidence of developmental C. mediterranea in Europe (see Fig. 2 in Noh & Henry, 2010) plasticity affecting song traits. For species after species, we and of C. plorabunda, C. adamsi, C. johnsoni,andC. downesi in have recorded the songs of the conspecific progeny of gravid North America (Henry, 1985a; Wells & Henry, 1992b;Henry females collected from locations thousands of kilometers & Wells, 2004, 2010). The results are highly consistent. First, apart, at high and low elevations, in every season of the year, each species responds nearly exclusively to its own song and and across different decades. We have reared additional does not answer the song of the other taxon (typical result progeny on several different diets and under long and short shown in Fig. 8, C. adamsi versus C. plorabunda). Second, males photoperiods, cold and warm conditions, and highly variable ◦ and females of each species discriminate equally strongly humidity regimes, prior to recording their songs at 25 C. against the songs of non-conspecifics, that is neither sex In no cases (except those few mentioned earlier showing is more likely than the other to make mistakes (a possible genetically based clinal variation) did measurements of songs exception is C. agilis, whose males are significantly less choosy taken from conspecific individuals of such varied origins and than females; see Fig. 2A in Noh & Henry, 2010). Third, backgrounds differ by more than a few percentage points. any incorrect responses made to the heterospecific song type are usually concentrated at the beginning of an interaction and are not delivered in the normal iterative duetting format. VI. REPRODUCTIVE ISOLATION Translating these laboratory results to the field, it is clear that distinctive duets are almost certain to stop courtship between individuals of different species, preventing copulation and (1) Premating, prezygotic isolation the production of hybrids. Sympatry frequently occurs between the widespread species (2) Postmating, postzygotic isolation comprising the carnea-group. For instance, C. agilis, C. lucasina, C. pallida, C. carnea,andC. mediterranea all co-occur in the When premating barriers between species have been southern Swiss Alps (Noh & Henry, 2010), while C. adamsi, circumvented by prolonged confinement in a small C. johnsoni, C. ‘downesi-2’, C. downesi,andC. calocedrii live laboratory arena, fertilized females readily produce healthy together in the San Bernardino Mountains of southern first-generation hybrids that in turn can produce second- California (Henry et al., 2012). At many sites in these regions, generation and backcross progeny. Contrary to the it is even possible to collect three or four different cryptic expectations of Haldane’s rule (Orr, 1997) under XY sex

Chrysoperla adamsi Chrysoperla plorabunda almost impenetrable barrier to interspeciﬁc reproduction. The combined presence of strong premating reproductive isolation but weak postmating isolation is one of the signs (volley period) (volley period) of rapid, recent speciation in a clade, especially when 1.0 coupled with low levels of genetic differentiation among N =13 N =12 its species (Coyne & Orr, 1997). Speciation, song genetics, 0.8 and phylogenetic relationships in the carnea-group will be

0.6 To own song addressed later in this review. To other song 0.4 *** 0.2 *** VII. ACOUSTIC SPACE AND SONG EVOLUTION Proportion of responses 0.0 C. adamsi vs C. plora C. plora vs C. adamsi In geographical areas occupied by sympatric species of Direction of interaction lacewings, duetting songs are highly distinctive, and because Fig. 8. Proportion of total responses by C. adamsi and they are distinctive, behavioural isolation among the cryptic C. plorabunda individuals to playback signals of their own song species is very strong. Thus, the five sympatric species that versus the song of the other species. N = number of individuals live in Europe occupy largely different regions of acoustic tested, asterisks indicate significant population differences space when the results of multivariate analyses of their song (***P < 0.0005), and error bars are standard deviations of the phenotypes are graphed (Fig. 9). The same is true of the mean. Oscillograms, drawn to the same time scale, compare songs of five sympatric North American species (see Fig. 5 typical duetting songs of C. adamsi and C. plorabunda.Methods in Henry et al., 2012). Even a superficial visual examination and data are in Henry & Wells (2010). of oscillograms and spectrograms will verify this disparity among the species confined to single continents (Fig. 3). It is likely that the existence of distinctive songs among determination (Sem´ eria,´ 1984), sex ratios in F hybrids are 1 sympatric species reflects the inability of two species having not female-biased, nor has evidence been found of hybrid similar songs to co-exist. The cause is not competition and male sterility. To date, successful hybridization has been competitive exclusion (see further discussion in Section VIII), achieved in four out of the ten possible interspecific crosses but simply fusion of the two taxa into a single entity through between the five taxa in Europe (Noh & Henry, 2010) and hybridization. The sympatric congeners C. plorabunda and in four out of the six possible crosses between C. plorabunda, C. adamsi provide a useful example of this phenomenon. C. adamsi, C. johnsoni,andC. downesi in North America Of all North American lacewings, those two species are (Tauber & Tauber, 1977; Henry, 1985a; Wells, 1993; Henry, the most similar to each other acoustically (discriminant Wells & Holsinger, 2002b; Henry & Wells, 2010). Similar success was achieved in two intercontinental crosses – the first between German C. carnea and Japanese C. nipponensis (Naka, Mitsunaga & Mochizuki, 2005; Naka et al., 2006), and 2 C. lucasina (N=152) C. mediterranea (N=57) the second between European C. pallida and North American C. carnea (N=64) C. calocedrii (Henry et al., 2012). C. pallida (N=52) 1 For two of the interspecific crosses listed above, careful C. agilis (N=72) counts were made of eggs laid, eggs hatched, larvae surviving, and adults eclosing. In those experiments, F1,F2,and 0 backcross generations exhibited a measurable decline in PC-2 fertility and fecundity, even though all categories of offspring generally survived and reproduced well. For example, in C. -1 plorabunda x C. johnsoni, hatching success was reduced by a modest 16% in the F1 generation and by an additional 15% -2 in the F2 generation, relative to conspecific control crosses (Wells, 1993). By contrast (but using different methods), Naka -3 -2 -1 0 1 2 et al. (2005) reported highly variable reductions in fertility in PC-1 C. carnea x C. nipponensis, with the amount of decline dependent Fig. 9. Scatterplot of the first two principal components of upon which species constituted the male or female parent. a principal components analysis (PCA) using 19 features of In summary, postzygotic isolation is present but relatively the songs of five cryptic, sympatric lacewing species found in weak among the species of the carnea-group, and it does Europe. Each data point represents a single individual, coded not obey Haldane’s rule. At the levels measured in the by taxon. N = number of individuals in each taxon; at least five experiments, it could not alone prevent the disappearance of songs were measured and averaged for each individual. Note most of these sympatric taxa if they were to hybridize with proximity of C. carnea and C. pallida in multidimensional acoustic each other. Instead, premating duetting songs constitute an space. For methods see Henry et al. (2012).

Biological Reviews 88 (2013) 787–808 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society 798 Charles S. Henry and others function and principal components analyses, see Fig. 5 in 275 Henry et al., 2012). To test whether there might be enough (A) = C. calocedrii acoustic space between them to accommodate a third species, 250 we produced ﬁrst-generation hybrids of intermediate song 225 phenotype as a surrogate for such a third species. This intermediate taxon (see Section V.2) did not show strong 200 behavioural discrimination against either C. plorabunda or C. adamsi, nor did it elicit much discrimination from those 175 species in return (see Fig. 5 in Henry & Wells, 2010). Thus the

Volley duration (ms) =C.pallida songs of C. plorabunda and C. adamsi occupy abutting regions 150 of acoustic space, and ‘limiting song similarity’ precludes 125 persistence of any song taxon that might occupy the acoustic space between them. 200 250 300 350

(B) (1) Parallel evolution 1400 = C. "adamsi-K" =C.adamsi By contrast, species that occupy different continents are not 1200 subject to limiting song similarity. Because the frequency range of tremulation signals in the carnea-group is narrow 1000 and entirely below 140 Hz, information content is also low

(measured by the required digital sampling rate), and this 800 places an upper limit on the number of truly distinctive Volley duration (ms) songs that can evolve. Consequently, over time, functionally 600 similar songs are likely to appear more than once, simply by chance. When this occurs within one continent, the fate of the 400 duplicate song type will be extinction through hybridization, 2000 2500 3000 3500 4000 4500 but when similar song types appear by parallel evolution 900 on different continents where mating interactions are not (C) possible, both instances of the song can persist. In the 800 carnea-group, three examples of such parallel song evolution 700 have been identiﬁed so far between North America and

Eurasia: C. adamsi and C. ‘adamsi-Kyrgyzstan’ (Henry, Wells 600 = C. plorabunda & Simon, 1999b), C. calocedrii and C. pallida (Henry et al., 2012), and C. plorabunda and ‘CcX-Asia’ (present review). For 500 each species pair, parallel evolution is supported by the joint 400 = "CcX-Asia" presence of song similarity and mutual song acceptability in Volley duration (ms) two taxa that are not their own closest relatives (mtDNA 300 sequences, Henry et al., 2012). Figure 10 illustrates the extent 200 of song convergence by plotting volley duration against volley 600 800 1000 1200 1400 1600 1800 2000 2200 period, two important temporal features that mediate species Volley period (ms) recognition within the carnea-group (Wells & Henry, 1992a; Henry & Wells, 2010). The ﬁrst two species-pairs (Fig. 10A, Fig. 10. Plot of volley duration at midpoint of the shortest B) show extensive overlap on both axes of the graph, while repeated unit (SRU) versus volley period at mid-SRU of the the third (Fig. 10C) shows overlap of volley period but songs of three pairs of species of the carnea-group exhibiting parallel song evolution between different continents. Each data not duration. Nonetheless, playback experiments (using 15 point represents the mean for a single individual, coded by taxon. individuals; C. S. Henry, unpublished data) conﬁrm that Oscillograms, all drawn to the same time scale, compare the ‘CcX-Asia’ cannot discriminate between its own song and typical duetting songs of all six species, i.e. (A) North American that of its North American analogue, C. plorabunda, implying C. calocedrii versus European C. pallida, (B) North American that functional convergence between continents does not C. adamsi versus Asian C. ‘adamsi-Kyrgyzstan’, and (C) North require that song phenotypes be identical. American C. plorabunda versus Asian ‘CcX-Asia’.

(2) Movement between continents weak, any movement of a species onto a new continent Ongoing evolution to ﬁll unoccupied song space is an engine already occupied by a species with a similar song would of speciation in the carnea-group (see Section VIII), but result in interspeciﬁc hybridization and disappearance of parallel evolution could cause reverse speciation (Seehausen, one of the species in the new region of sympatry. For van Alphen & Witte, 1997; Taylor et al., 2006) under certain example, if C. pallida from Europe were to disperse or conditions. Because postzygotic reproductive barriers are so be transported to southern California, it would readily

Biological Reviews 88 (2013) 787–808 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society Duetting and evolution in lacewings 799 hybridize with C. calocedrii. Depending on specific genetic hemispheres (Henry et al., 2012). Species that co-occur within and ecological factors, C. pallida or C. calocedrii might absorb continents maintain reproductive isolation by occupying its acoustic twin or (less likely) fuse into a new entity functionally distinct regions of acoustic space, delineated characterized by an intermediate phenotype. The unique by unique duetting songs. Allopatric species, on different features of C. calocedrii – its morphology and colour patterns, continents or in different hemispheres, can (and occasionally ecophysiology, song details, and even its DNA sequences – will) exhibit parallel song evolution. would be lost or muddled. Had the species not been described already (Henry et al., 2012), no-one would know it was gone. The live lacewing trade has taken intercontinental VIII. DUETTING AND SPECIATION transplantation of species from mere conjecture to reality. It has been proven that insectaries serving the biological control The carnea-group is a young, relatively speciose evolutionary industry have inadvertently sold a North American lacewing lineage that is rapidly diversifying. Interpretation of this species, C. plorabunda, in Europe; a European species, C. ongoing process of speciation requires careful consideration lucasina, in the southwestern U.S.A.; and another European of three independent characteristics of the clade: obligatory species, C. carnea, in both the U.S.A. and Japan (Mochizuki duetting prior to mating, simple genetic control of song & Mitsunaga, 2004; Naka et al., 2006; Henry & Wells, phenotype by segregating units of major effect, and 2007; Mochizuki et al., 2007, and C. S. Henry, unpublished remarkable genetic similarity among all cryptic species. data). These insectary-reared specimens are mass-released into agricultural fields, so the chances for establishment on (1) Three critical properties of the clade the new continent are probably high. The consequences of naturalization are difficult to predict. Only Mochizuki & Mitsunaga (2004) and Naka et al. (2005, 2006) have addressed (a) Duets enforce reproductive isolation these issues experimentally, concluding from the observed Individuals in nature will mate only if they can establish low rates of interspecific mating in large cages that native prolonged duets with their partners. Both sexes exercise C. nipponensis would probably maintain its integrity even if mate choice equally (e.g. Noh & Henry, 2010), through C. carnea were to become common in Japan. However, it is the mutually exchanged substrate-borne vibrational duetting important to note that C. carnea and C. nipponensis have highly song. These songs are complex, sexually monomorphic, distinctive duetting songs (Fig. 3); if such were not the case, and species-specific, and they constitute powerful premating a different outcome might be expected. reproductive barriers between species. In comparison, The magnitude of natural, non-anthropogenic dispersal postmating reproductive isolation between existing species of carnea-group species from one continent to another is is very weak throughout the clade. It follows that a change not known. As argued earlier, such events will not even in the mating song within a local population could isolate be knowable unless the song of the migrant differs from that population reproductively from all other members of those of all residents it encounters. Only one example has the original species, so duetting is likely to play a major role been verified, in which central Alaska has been colonized in speciation. Other potentially complicating issues such as by C. agilis from Eurasia (Henry et al., 2011). In this case, correlated evolution of signal and preference may be less almost 9000 km separate the Alaskan population from the critical in this system than others (e.g. Ritchie, 2007; Wiley nearest known Eurasian population (Fig. 4). Nevertheless, & Shaw, 2010), because males or females with a new song natural dispersal is a more likely explanation than recent will be predisposed to duet, and mate, largely with partners translocation by humans, because the population has that also sing the new song. persisted in Alaska long enough to become abundant and well adapted to extreme temperatures and photoperiods. The alternative hypothesis of parallel evolution can be rejected (b) The genetic basis of song differences is simple on two grounds. First, the chance of independent origin of Pairwise genetic crosses between several species have the entire suite of features characterizing the complex song shown that critical phenotypic differences in the songs are of C. agilis is very low. Second, Alaskan C. agilis clusters controlled by relatively few segregating units, each of major phylogenetically with Eurasian C. agilis, not with the North effect (Henry, 1985a;Henryet al., 2002b). Such type II American cryptic species (Henry et al., 2011); we would genetic architecture (Templeton, 1981), which is relatively predict North American affinities for the Alaskan population uncommon for behavioural isolating traits (e.g. Ellison, if it had arisen from a pre-existing species on that continent. Wiley & Shaw, 2011; Oh et al., 2012; Ritchie, 2000; Ting, Summarizing our current understanding, all studies Takahashi & Wu, 2001), could accelerate establishment of a suggest that lacewing songs have diversified primarily within newly arisen song phenotype in a lacewing population. More continents, as part of the process of speciation. Particular song specifically, in conjunction with the reproductive isolating phenotypes are strongly associated with DNA haplotypes, effect of a new duetting song, type II architecture could and these haplotypes cluster together phylogenetically into facilitate the process of speciation by allowing immediate, distinct clades largely confined to major continents or global unambiguous expression of a new song allele in a subset of

Biological Reviews 88 (2013) 787–808 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society 800 Charles S. Henry and others the population rather than dilution of song differences by and preference are phenotypically identical between males multi-locus recombination. and females of a species and almost certainly share the same genetic architecture in both sexes. Nonetheless, song (c) Cryptic phenotypes have cryptic genotypes variants must arise and become established for speciation to occur, and it is not clear how a new (large-effect) song Levels of genetic differentiation among the valid species of mutation could spread to enough individuals to create the group are very low. Jukes-Cantor corrected distances a reproductively isolated population. Possibly, geographic (J-C, see ch. 11 in Swofford et al., 1996) range from 0.005 heterogeneity facilitates speciation by physically confining to 0.027 for mitochondrial pairwise sequence divergence an individual bearing the mutation to a space occupied by across the entire clade (Henry et al., 2012), while Nei’s only one or two partners (Henry, 1985b). If ‘no choice’ genetic distances D (Nei, 1987) calculated from allozyme mating were then to occur and the numerous progeny data vary between 0.02 and 0.13 for selected species-pairs of the mutant cross were to remain cut off from the from Europe and North America (Cianchi & Bullini, 1992; main population for just one more generation, multiple Wells, 1994). These are values typically seen among local individuals homozygous for the new allele will appear. Such populations, not species, of other insects (e.g. butterflies, individuals will express a highly distinctive song phenotype, Nice & Shapiro, 2001). Even the Hawaiian cricket genus causing strong assortative mating that isolates bearers of Laupala, characterized by ‘ ... the highest rate of speciation the homozygous genotype. This hypothesis is consistent so far recorded in arthropods’ (Mendelson & Shaw, 2005, with existing geographic patterns of species diversity in the p. 375), exhibits J-C mitochondrial sequence divergences carnea-group: the largest number of co-occurring species of 0.026–0.040 (Shaw, 1996) – larger than found in the is always found in topographically rugged mountainous carnea-group. This evidence of genetic similarity, together regions such as the Sierra Nevada of California, the Alps with the presence of strong premating and weak postmating of Switzerland, the Caucasus of Armenia and Georgia, and reproductive isolation, indicate that the carnea-group, too, the Tien Shan of Kyrgyzstan (Wells & Henry, 1998, and has recently undergone rapid speciation. Recent, rapid Fig. 4). Around these mountain massifs, glacial cycles are speciation is also an important source of ambiguities in the likely to have repeatedly created isolated populations in Bayesian phylogenetic tree (Fig. 11), here inferred from 4630 refugial zones. Fossil evidence from assemblages of insects base-pairs of hypervariable mitochondrial DNA from four and other biota suggests that there has been limited if any protein-coding genes (Henry et al., 2012). Although many speciation or extinction throughout the Quaternary, due nodes are well supported on this tree, several taxa appear to remixing of populations when they emerge from refugia on more than one branch, including C. agilis, C. mediterranea, after each glacial-interglacial cycle (Bennett, 2004; Coope, C. plorabunda, and especially C. nipponensis.Furthermore,all 1995); it is thought that such remixing effectively removes Eurasian species arise from very short branches and do not any microevolutionary developments. In the carnea-group, even constitute their own monophyletic assemblage. Such the species persist because the songs isolate the species even inconsistent patterns are not unexpected among recently when they remix following the glacial or interglacial period. formed species, especially if they are distinguished almost entirely by behavioural traits (duetting songs) encoded by (3) Ecology and speciation relatively few genetic elements of large effect (type II architecture). Speciation in the absence of ecological differentiation is contrary to accepted theory, despite its likelihood in the carnea-group. Competitive exclusion should prevent closely (2) Mode of speciation related species from occupying identical ecological niches The actual process of speciation in the carnea-group remains (Hardin, 1960). Also, recent surveys of speciation suggest unknown. Ecological (adaptive) speciation, at least with that the primary correlates of cladogenesis are likely to respect to the evolutionary responses of songs to distinct be ecological in most organisms (Rundle & Nosil, 2005; substrate properties, has been shown to be unlikely (Henry & Schluter, 2009; Sobel et al., 2010; Weissing, Edelaar & Wells, 2004). On the other hand, speciation via mutual sexual Van Doorn, 2011). However, several cryptic lacewing selection, in which both sexes are choosy, seems plausible. species occupy the same locality at the same time and A hypothetical variant of such a process has been explicitly eat at least some of the same foods. Such syntopy in modelled by Almeida & de Abreu (2003). In the model, a new other taxa is typically associated with enhanced rather secondary sexual trait arises in either sex of a population, than low character divergence, produced by the combined inherited as a dominant allele at a single locus; the new processes of ecological character displacement, ecological genotype is selected as a mate largely by other individuals speciation, and taxonomic extinction (Dayan & Simberloff, that also bear the allele, leading to disruptive sexual selection 1996). Seehausen & van Alphen (1999) provide a possible and non-allopatric divergence through a sequential, mutual solution to this dilemma. The haplochromine cichlid fishes mate-matching process (Puebla, Bermingham & Guichard, of East Africa, like the lacewings, constitute a swarm of 2011). It is easy to see how the carnea-group might fit this species showing little ecological differentiation. Focusing model. In fact, the modelled process would work even more on modes of speciation, Seehausen & van Alphen (1999, effectively in lacewings than in other animals, because signal p. 262) proposed that divergence via sexual selection is so

C. z. sillemi (India & Middle East) C. z. zastrowi (South Africa)

1 C. lucasina (Switzerland)

C. nipponensis “A2” (Japan)

C. agilis (Alaska & Switzerland)

.79 C. lucasina (southern Europe)

C. mediterranea (Greece)

.80 1 C. pallida (France, Switzerland, & Greece) C. renoni (Hungary)

C. agilis (Switzerland) .86 1 C. mediterranea (Pfynwald of Switzerland)

Eurasian/African species C. “adamsi-Kyrgyzstan” (central Asia) 1 .69 C. agilis (Alaska & Switzerland)

.77 C. carnea (France, Switzerland, & Finland)

.99 1 C. nipponensis “A1” (Japan) C. “nipponensis B” (Japan) 1 C. calocedrii (southern California)

1 1 C. “downesi-2” (northwest USA) C. downesi (northeast USA) 1 C. plorabunda (Moscow, Idaho) 0.03 1 1 C. plorabunda, adamsi,& johnsoni 1 C. d. “mohave” (southern California)

N. American spp. 1 C. adamsi, johnsoni,& d. “mohave”

1 C. comanche 1 (North America) C. rufilabris C. pudica (Africa) Fig. 11. Bayesian inference (BI) phylogram of the cryptic species of the carnea-group, based on 4630 bp of mitochondrial sequence from the protein-coding genes ND2, COI, COII,andND5. The BI procedure was implemented using a GTR + + I model, applied to each of the four gene partitions and with all parameters unlinked. Nearly identical topology and branch lengths were obtained in a maximum-likelihood analysis (ML) using the same substitution model. Numbers at the branch points are Bayesian posterior probabilities; branch lengths are measured as the number of substitutions per site (horizontal bar). Modified and redrawn from Henry et al. (2012), where complete methods can also be found. rapid in the Rift Valley Lakes that the rates of speciation song phenotype between populations is greater in sympatric ‘ ... outrun rates of extinction by competitive exclusion or than allopatric regions (Brown & Wilson, 1956; Butlin, 1987; divergence by character displacement ...’. Like the cichlids, Schluter, 2000). Although RCD has been found in many carnea-group lacewings are also undergoing recent, rapid other acoustical insects (Hewitt, 1990; Ritchie, Butlin & speciation – perhaps fast enough to have similarly outpaced Hewitt, 1990; Benedix & Howard, 1991; Marshall & Cooley, ecological processes that normally suppress such occurrences 2000; Massie & Markow, 2005; Jang & Gerhardt, 2006; (‘eco-evolutionary dynamics’, reviewed by Schoener, 2011). Popple, Walter & Raghu, 2008), it has not been confirmed in the carnea-group, despite an ongoing search. A continentally sympatric pair of highly vagile species rarely presents clear (4) Does reinforcement complete speciation? regions where allopatry can be proven (Fig. 4), so at the onset Reproductive character displacement (RCD), a signature of it is difficult to choose suitable populations for comparison. possible reinforcement, can be inferred when divergence of Also, the songs of the species being compared in each pair

4.0 and the better we understand their morphology, ecology,

3.5 C. adamsi and distribution, the more reliably we can interpret and 3.0 assess the work of other research groups published under the 2.5 C. carnea name (Duelli, 2001). adamsi adamsi 2.0 (S. California) (Oregon) As is often true of cryptic species, identifying reproductively isolated units within an otherwise homogeneous complex 1.5 C. plorabunda of individuals has been the first step toward discovering 1.0 plorabunda plorabunda previously unnoticed physical and ecological features Volley/SRU period (s) 0.5 (Connecticut) (Oregon) distinguishing members of the carnea-group. Subtle and 0.0 seemingly random differences often become more obvious Allopatric Sympatric 12 s and make more sense when they can be associated with Fig. 12. Box plots (showing median, 25th/75th, 10th/90th, and song-delimited taxonomic entities. Duelli, Obrist & Fluckiger 5th/95th percentiles) of volley or shortest repeated unit (SRU) (2002) demonstrated this principle in their study of species- period in allopatric versus sympatric populations of C. adamsi specific differences in adult morphology, habitat association, and C. plorabunda in North America. The regions of sympatry and seasonal migration in sympatric Swiss populations of and allopatry are specified beneath each box plot. Numbers the cryptic species C. carnea, C. pallida,andC. lucasina. of individuals measured in each class included 37 allopatric plorabunda, 30 sympatric plorabunda, 23 allopatric adamsi,and15 In a second example, distinct larval colour patterns were sympatric adamsi. Oscillograms of the two songs are shown to found to be clearly linked to duetting song differences the right of the graph. between the recently recognized species C. nipponensis and C. ‘nipponensis-B’ (Taki, Kuroki & Nomura, 2005; Haruyama et al., 2008; Henry et al., 2009; Mochizuki must resemble each other in basic volley and SRU structure, et al., 2010). which is not usually true in the carnea-group. Working within Adults of most species of Chrysopidae can be recognized these limitations, studies have explicitly failed to find RCD by unique morphological characteristics of the male genitalia in allopatric-sympatric song comparisons of C. carnea, C. (Tjeder, 1966). However, while male genitalia are useful in mediterranea and C. pallida in Europe (Henry et al., 2002b)and distinguishing several species of Chrysoperla (Brooks, 1994), C. plorabunda and C. adamsi in North America (see Fig. 12 most species in the carnea-group share very similar genitalic and Henry & Wells, 2010). It should be noted, however, that morphology (Henry, Wells & Pupedis, 1993). Nevertheless, these studies did not test for RCD that may be present as there is significant morphological variation within and different preferences for the signal, rather than as divergent between carnea-group species in, for example colour of the phenotypes of the signal itself (Higgie & Blows, 2007; Noh & head, thorax and wing veins, the distribution of black or Henry, 2010). blond abdominal and thoracic setae, and the extent of In general, these results suggest that songs in the carnea- the basal dilation of the tarsal claw. This has led some group have probably not diverged in response to the presence authors to use these characters to designate species limits. of other song types within a geographical area. We suggest that two sympatric songs are either sufficiently distinct to Unfortunately, a morphological approach is likely to give begin with, or the populations possessing them have merged spurious results and can lead to circularity unless the through hybridization to create a single song phenotype. If distribution of such characters is tested against independent instead reinforcement and concomitant RCD were major data, such as courtship songs (see Section X). When carnea- forces in the divergence of these rapidly evolving species, group populations are segregated on the basis of their one would expect to find at least a few instances in nature courtship songs it becomes clear that while one species of active hybrid zones, where selection against intermediate may possess a unique morphological feature, another species phenotypes is in the process of strengthening the reproductive may exhibit more than one state in the same character (e.g. barriers between populations. Work by several researchers claw basal dilation in C. zastrowi versus C. mediterranea;see over many years has not located a single such hybrid zone. Henry et al., 1999a, 2006). The mechanisms generating species diversity in the carnea- Subtle morphological differences may help entomologists group are more likely to operate in some other manner (see to identify similar, closely related species, but the lacewings Section VIII.2). themselves do not use any such characters when they choose their own sexual partners. Although the extent of morphological divergence among populations may sometimes indicate genetic distances and hence phylogenetic IX. THE IMPORTANCE OF MORPHOLOGY relationships, the absence of such morphological differences does not necessarily indicate that just one species is present. Thus far, we have focused largely on the duetting songs About half of the song-derived species are truly cryptic, of the carnea-group. However, when viewed within a even when subjected to critical morphological examination. behavioural context, morphology can also contribute to However, even individuals of truly cryptic species can our understanding of the evolution and classification of these correctly identify their conspecific partners through exchange cryptic species. In fact, the more cryptic species we recognize of courtship songs.

As in the adults, much of the morphological variation Another research group also challenged the use of seen in larvae of the carnea-group is intraspecific rather than song phenotype for delineating the boundaries between diagnostic of distinct taxa (e.g. Henry et al., 2002a). However, carnea-group species. In a recent study, Thierry et al. there are exceptions. For example, Mochizuki et al. (2010) (2011) measured four morphological characters in 325 documented consistent differences in melanization of the European adults collected at 14 sites from the Azores in head capsule in C. nipponensis versus C. ‘nipponensis-B’ in the west to Poland and Bulgaria in the east. The authors Japan. Also, Californian C. calocedrii is as unique in its larval then present evidence from morphometric, ecological, and stages as it is in adulthood: it has the only larva in the DNA data supporting hypotheses of ecological character carnea-group characterized by white rather than greyish-tan displacement, reinforcement, and incipient speciation in background pigmentation over most of the body (Henry et al., morphological species that they call ‘affinis’ and ‘carnea’ 2012). (Fig. 13). Unfortunately, this study tested hypotheses that In summary, the species of the C. carnea-group display a were based on the assumption that morphology is highly potentially confusing array of morphological features which informative and stable in the carnea-group, even though in some species or populations provide diagnostic characters, neuropterologists – including one of the authors of the while in others are not suitable for reliable identification. study – have long known it is not (Smith, 1932; Tjeder, Morphology can, however, provide a useful supplementary 1960; Thierry & Adams, 1992; Brooks, 1994; Henry et al., means of identifying carnea-group species, but only after a 2001). When song-delineated species are correctly substituted species has been diagnosed using courtship song analysis. for Thierry et al.’s (2011) morphologically identified entities (Fig. 13), the conclusions are very different. Rather than finding character displacement and incipient speciation in carnea X. DENYING SONG-DELINEATED SPECIES: the -group, Thierry et al.’s (2011) results simply confirm STUDIES FROM THE LITERATURE that there are four reproductively isolated and previously described species present in Europe: C. pallida, C. agilis, C. carnea,andC. lucasina.InnorthernEurope,C. carnea coexists The validity of song-delineated species in the carnea-group with C. lucasina and C. pallida, whereas in southern Europe, has received extensive support not only from behavioural C. carnea is replaced by C. agilis in the species assemblage observations and experimentation, but also from traditional (Fig. 4). Where C. carnea and C. agilis overlap (southern Alps morphology and ecology. However, some authors have and southern Caucasus), all four species co-occur and come chosen to view duetting songs in the carnea-group as to the same lights at night (P. Duelli, unpublished data). No highly polymorphic traits within morphologically delineated song hybrids have ever been found in nature. In this case, species, without special significance as species-specific ecological character displacement is not providing a ‘route markers. to speciation’, as suggested by Thierry et al. (2011); instead, One such study focused on phenotypic plasticity in the speciation has already occurred through other mechanisms ‘carnea complex’ (Tauber & Tauber, 1992; Tauber et al., and is now a fait accompli. 2000). The authors proposed the existence of a single species, ‘Chrysoperla carnea’, in western North America, comprising many populations that ‘exhibit considerable geographic variation’ (Tauber & Tauber, 1992, p. 1757). They interpreted the ubiquity and unordered nature of this variation to mean that the taxon could not be divided C. lucasina “carnea” “affinis-a” “affinis-s” into more than one species. Instead, they hypothesized the Thierry et al. (2011) existence of broad phenotypic plasticity in the life-history traits of ‘Chrysoperla carnea’, controlled by population-specific C. lucasina C. pallida C. agilis C. carnea Relative mtDNA divergence developmental switches activated by photoperiod and food Present review availability. We know now that as many as six distinct taxa potentially co-occur where these authors collected their Fig. 13. Diagram summarizing the differences between Thierry specimens: C. plorabunda, C. johnsoni, C. adamsi, C. downesi, et al.’s (2011) hypothesis of intraspecific character displacement C. ‘mohave’, C. ‘downesi-2’, and C. calocedrii.Thusatleast and a null hypothesis (present review) that assumes stable co- some of the ‘geographic variation’ and ‘phenotypic plasticity’ existence of three valid song-delineated species in the European described by Tauber & Tauber (1992) was probably carnea-group. Note that ‘carnea’ of Thierry et al. (2011) is actually C. pallida, while their incipiently speciating ‘affinis’ is C. agilis associated with those distinct taxonomic units, rather in the southern part of its range and C. carnea in the northern than being distributed across ‘C. carnea’inadisorganized part. The fourth species, C. lucasina, is sympatric with the other patchwork. Moreover, the genetic crosses described in their three species throughout their ranges. Mitochondrial DNA paper certainly included interspecific pairings, confounding divergence (vertical axis) reflects relative p distances among their results further. Correctly identifying the song phenotype populations taken from Fig. 5 in Thierry et al. (2011). Song of each specimen used in experiments such as these is the oscillograms are shown below each valid species name. As in only way to understand the observed patterns of life-history Fig. 3, the arrows mark where the partner in the duet inserts its variation across western North America. SRU (= song).

XI. CRYPTIC SPECIES, SYSTEMATICS, AND exhausted the potential of mtDNA, we must now include BIOLOGICAL CONTROL rapidly evolving nuclear DNA sequences and nuclear markers for phylogenetic inference. From a well-resolved, The importance of applying correct taxonomy to both stable phylogeny, patterns of character evolution and rates the agents and targets of biological control programs of speciation within the clade can be inferred accurately. has been recognized for a long time (Clausen, 1942). In Such a tree will also permit better tests of hypotheses more recent years, cryptic species have received increasing of parallel and convergent song evolution, dispersal versus attention, especially as DNA analyses, morphometric studies, human translocation of taxa, and divergence of multiple and ecophysiological experiments have revealed hidden ecotypes within single-song species (e.g. the ‘mohave’ ecotype taxonomic diversity within species previously thought to be arising independently within C. downesi and C. johnsoni;see undifferentiated, e.g. numerous parasitoid wasps (Nagarkatti, Henry, 1993b). 1975; Stouthamer et al., 2000; Triapitsyn et al., 2010) and Future studies should also address the population beetles that feed on invasive plants (Davis et al., 2011; Tosevski genetics of the species complex. The Bayesian phylogram et al., 2011). Although the cryptic species of the carnea-group (Fig. 11) shows several instances where two distinct species are not host-specific like the examples above, it is nevertheless share the same mitochondrial haplotype or possibly the equally important that lacewings are correctly identified. same mitochondrial genome. Phylogeographic analysis Certain species are associated with certain habitats, e.g. will determine whether these cases represent ancestral deciduous trees for C. pallida, herbaceous vegetation for polymorphisms, incomplete lineage sorting, or mitochondrial C. agilis, C. carnea,andC. plorabunda, and evergreens and capture due to interspecific hybridization (Spooner & conifers for C. mediterranea, C. calocedrii,andC. downesi. Thus, Ritchie, 2006; Knowles & Chan, 2008; Polihronakis, 2010). it is unlikely that a lacewing species will effectively control General estimates of gene flow and introgression in the resident arthropod pests if released into unsuitable habitats group will also be useful. Based on the presence of a stabilized (Pappas, Broufas & Koveos, 2011). duetting song in each species, we tend to assume that levels of Indiscriminate translocation of living lacewings across vast such hybridization are very low; however, the mitochondrial distances has been and is still practiced by small and large tree possibly tells us otherwise. For example, hybridization commercial insectaries alike, reflecting a persistent belief that may be relatively common, but not obvious because of strong the carnea-group is a single polymorphic species (Tauber et al., stabilizing selection on the ‘speciation genes’ controlling song 2000). This has brought shipments of North American species phenotype (Wu, 2001; Orr, Masly & Presgraves, 2004; Nosil to Europe and European species to both North America and & Schluter, 2011). Asia (see Section VII.2), although as yet there is no evidence Ultimately, we should undertake genetic mapping studies that non-native species have undergone naturalization after of carnea-group species and develop genomic resources for importation (Henry & Wells, 2007; Mochizuki et al., 2007). them, as is currently being done in other non-model systems Three problems can result from incorrect taxonomy. First, such as Timema stick insects (Comeault et al., 2012) and there is the obvious waste of resources that accompanies Laupala crickets (Ellison et al., 2011). Discovery of quantitative a control initiative using a species that is inappropriate for trait loci (QTLs) and candidate genes associated with the chosen crop or forest environment. Second, an exotic critical song differences between interfertile taxa will permit control agent could hybridize with a native species that sings identification of ‘song genes’ in the carnea-group, analogous similarly, eliminating both species as originally delineated to the period gene in the Drosophila buzzatii complex (Franco and destroying the special adaptations that each might have et al., 2010). If songs are indeed the speciation trait, as to its original environment. Third, an introduced species seems likely, then characterizing the genetic architecture might competitively displace one or more native species in of song phenotype and mate preference in these lacewings a particular environment. Again, this is a highly negative will provide important insights into the poorly understood outcome, from a conservation biology perspective. The processes responsible for the rapid proliferation of cryptic effort should be made to identify cryptic species accurately, species in lineages characterized by strong sexual selection taking care to use only local species as agents of biological (e.g. Otte, 1989; Schiffer, Carew & Hoffmann, 2004; control in different geographical regions. This should not Mendelson & Shaw, 2005; Pierotti & Seehausen, 2007; present serious problems, because duetting songs are easily Sueur & Puissant, 2007; Pauers, 2010; Pennetier et al., 2010; observable and at least one member of the carnea-group can Marshall et al., 2011). be found near almost every agricultural site. As we have indicated, there are both commercial and ethical rewards for practicing responsible behaviour. XIII. CONCLUSIONS

(1) Many aspects of the biology, evolution, and systematics XII. FUTURE DIRECTIONS of the Chrysoperla carnea-group of green lacewings (Neuroptera, Chrysopidae) have been profoundly shaped by their A primary goal of future research is to produce a robust unique substrate-borne duetting songs, which are produced phylogeny for all taxa in the carnea-group. Having perhaps by vigorous vibration of the abdomen. These sexually

Biological Reviews 88 (2013) 787–808 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society Duetting and evolution in lacewings 805 monomorphic, low-frequency courtship signals must be XIV. ACKNOWLEDGEMENTS exchanged reciprocally between individuals before they will mate. This review spans a research program of many years, so we (2) What has until recently been considered a single cannot name everyone and each organization that helped globally distributed morphological species, Chrysoperla carnea, make the work possible. However, we can thank our current is now known to comprise at least 21 distinct cryptic species. institutions, specified on the title page, for their logistical Each has its own species-specific duetting song and an and financial assistance, and various granting agencies (e.g. extensive geographic distribution, often spanning thousands the National Science Foundation and U.S. Department of linear kilometers. Within a continent, species are usually of Agriculture) for supporting some of our field work and broadly sympatric. Songs are characterized by a wide range experiments over the years. More specifically, we are grateful of temporal and spectral qualities, constrained only by their to Drs Suegene Noh (Kansas State University) and Chris restriction to very low frequencies. Simon (University of Connecticut) for constructive criticism (3) Phenotypic variation of songs within each species is of the manuscript, and to two anonymous reviewers for typically very low, although a few widely distributed taxa improving its clarity and focus. show clinal variation. In addition, some degree of song plasticity enables partners to synchronize their signals quickly during duets. In a few species, plasticity permits males and XV. REFERENCES females to tell each other apart. 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(Received 24 April 2012; revised 18 January 2013; accepted 25 January 2013; published online 22 February 2013)