The evolutionary loss of sexual traits is widely predicted. Because sexual signals can arise from the coupling of specialized motor activity with morphological structures, disruption to a single component could lead to overall loss of function. Opportunities to observe this process and characterize any remaining signal components are rare, but could provide insight into the mechanisms, indirect costs and evolutionary consequences of signal loss. We investigated the recent evolutionary loss of a long-range acoustic sexual signal in the Hawaiian field cricket Teleogryllus oceanicus . Flatwing males carry mutations that remove sound-producing wing structures, eliminating all acoustic signalling and affording protection against an acoustically-orientating parasitoid fly. We show that flatwing males produce wing movement patterns indistinguishable from those that generate sonorous calling song in normal-wing males. Evolutionary song loss caused by the disappearance of structural components of the sound-producing apparatus has left behind the energetically costly motor behaviour underlying normal singing. These results provide a rare example of a vestigial behaviour and raise the possibility that such traits could be co-opted for novel functions.

1. Introduction

The evolutionary loss of sexual signals is a central prediction of sexual selection theory [1]. Its widespread occurrence is supported by numerous examples inferred from phylogenetic studies, but the rarity of contemporary cases makes it challenging to study its evolutionary dynamics [2]. Since sexual signalling frequently involves the coupling of multiple trait components, such as complex motor activities and specialized morphologies [3], its evolutionary loss might be predicted to occur in a stepwise fashion. If initially only one component is lost, others may be left behind as non-functional vestigial traits [4]. Characterizing such vestiges could help reveal evolutionary constraints or paths of least resistance leading to trait loss, fitness consequences of trait loss, and mechanisms by which vestigial traits might be co-opted for new functions [5].

We addressed this by studying field crickets (Teleogryllus oceanicus) that experienced the recent evolutionary loss of male song. Males sing to attract females for mating, but in Hawaii their song also attracts female parasitoid flies (Ormia ochracea) whose larvae burrow into, consume and kill their host [6]. A novel, genetic male morph incapable of producing song (flatwing) was discovered in 2003 on the island of Kauai [7]. Currently, approximately 95% of males on Kauai and 50% of males on the neighbouring island of Oahu express the flatwing phenotype [7,8]. Crickets normally produce acoustic signals by rhythmically opening and closing their forewings, scratching the scraper of one wing against the file of the other [9], but these wing structures are severely reduced or absent in flatwings (figure 1a) [7,8]. We used an opto-electronic camera [10] to test whether flatwing males continue to express the stereotyped wing movements that produce sonorous calling song in normal-wing males, and if they do, whether wing movement patterns differ between morphs. Figure 1. (a) Structural differences in the forewings of normal-wing and flatwing males. Flatwing males lack, or have severely reduced, vibration-generating (file and scraper) and resonating structures (mirror and harp). Yellow symbols indicate the placement of reflective markers used for opto-electronic measurements of wing movement. (b) Vertical forewing movements associated with singing. (c) Representative calling song from a normal-wing male (top), with corresponding wing-movement recording (bottom). We measured 16 wing-movement parameters corresponding to key song components (described in the electronic supplementary material, table S1). Components fall into three categories: numbers of chirps or pulses (1–3), long-duration features on the order of seconds (4–7) and short-duration pulse or interval traits on the order of milliseconds (8–16). (d) Enlarged section of song from (c).

2. Material and methods

(a) Cricket origins and husbandry

Homozygous flatwing (n = 6) and normal-wing (n = 6) lines of T. oceanicus were established in 2012 from stock populations originating from Oahu, using the crossing methods detailed in Pascoal et al. [11]. Crickets were reared in 16 l plastic containers, at ca 25°C on a 12:12 hour light:dark cycle. We provided cardboard for shelter, cotton wool water pads and ad libitum access to Burgess Excel dwarf rabbit food. At least 4 days before experimentation, reproductively mature adult males were separated into single-sex containers.

(b) Experimental procedures and analysis

Wing movements were measured using an opto-electronic camera with a position-sensitive photodiode as a sensor, as described in [10] and figure 1. In this procedure, males with reflective markers adhered to their forewings are placed on a turntable in front of the camera and microphone. Red light is directed from the camera towards the cricket, and the reflection from the marker is used to measure wing position. During singing, males raise their forewings above the abdomen. The wings open and silently move downward to either side, and in normal-wing males they produce a sound pulse as they return upwards to close (figure 1b). During this process, the camera measures the vertical position of the marker as a voltage signal, which together with a microphone signal, is recorded by a PC running Spike2 software (CED, Cambridge, UK).

Wing movements associated with singing typically occur in bouts lasting several seconds to minutes. Before recordings took place, a reflective marker (3M Laboratories, Scotchlite foil type 7610, 1.0 mm diameter) was adhered to the costal margin of each male's forewing using PVA glue, and subjects were isolated in transparent 150 ml tubs. If we observed a subject attempt to sing during incidental visual monitoring, then it was selected for recording. The male was placed in front of the camera and microphone and the camera was adjusted to monitor its wing movements. The system only enabled recording one male at a time.

The number of recordings per cricket ranged between 1 and 13. We retained recordings for onward analysis if they contained at least 20 s of continuous singing, and we measured 10 consecutive songs from the earliest such bout of each recording. Of 52 crickets fitted with reflective markers, 16 individuals passed these criteria (9 flatwing, 7 normal-wing, from 9 different lines). Recording took place over 11 days, at 20°C under low light. We separately verified that flatwing males produce no sonorous signal up to ca 48 kHz (electronic supplementary material, figure S1).

Teleogryllus oceanicus calling song consists of two distinctive pulse patterns: the ‘long chirp’, containing a series of 5 to 8 pulses that typically increase in amplitude, followed by ‘short chirps’ (or trills), which are lower amplitude and contain multiple pairs of pulses [6]. Calling song can be further characterized by frequency and temporal components reflecting pulse durations and intervals. Figure 1c,d shows a simultaneous recording of the sound and corresponding wing movements produced during calling song, with 16 song components illustrated. We observed that both male morphs produce wing movements containing these components, so we then tested for quantitative differences between morphs.

We ran two general linear mixed models for each song component to test for differences between morphs. One model included ‘morph’ (flatwing/normal-wing) as a fixed effect, while the other did not. ‘Individual’ was always included as a random effect to account for the non-independence of within-subject recordings. A likelihood-ratio test was used to compare goodness of fit and assess evidence for variation in wing movements between morphs. Analyses were run using lme4 in R v. 3.2.4 [12].

3. Results

Both male morphs expressed the overall long chirp/short chirp pattern of wing movements (figure 2a) (electronic supplementary material, figure S2). Moreover, we found no significant quantitative differences between morphs for any of the 16 song components after correction for multiple comparisons (Bonferroni correction; k = 16, α = 0.003) (figure 2b). Without correction, there was a significant difference (p = 0.036) in component 16 (figure 1c; electronic supplementary material, table S1). The electronic supplementary material contains statistical details and video of a flatwing male moving his wings in a stereotyped calling-song pattern (electronic supplementary material, table S1 and video S1). Figure 2. (a) Five-second excerpts of wing-movement recordings for a normal-wing (top) and a flatwing (bottom) male illustrating that both morphs are capable of producing the distinctive two-part composition of the T. oceanicus calling song (a trill-like ‘long chirp’ followed by a series of lower amplitude ‘short chirps’). (b) Comparisons of wing-movement data between male morphs for individual song components, grouped and labelled with numbers as illustrated in figure 1c. Yellow dots, thick black bars and thin black lines indicate medians, inter-quartile ranges and 95% confidence intervals, respectively, and the shaded regions show probability density estimates for the data (grey: normal-wing males, red: flatwing males).

4. Discussion

The evolutionary disappearance of a sexual signal in T. oceanicus was caused by the loss of a key morphological trait, but we found that behavioural movement patterns underlying signal generation persist in high fidelity. Silent flatwing males continue to express the stereotyped wing motor behaviours produced by normal-wing males during calling song. This ‘silent singing’ provides a rare example of a vestigial behaviour [13] and affords three insights into the evolutionary dynamics of trait loss.

First, gradual evolutionary reduction of costly traits after selection ceases is a defining feature of vestigial characters [4], but we did not detect signs of such decay in patterns of wing movement during silent singing in T. oceanicus. In crickets, song-generating wing movements are energetically costly [14,15]. For example, the metabolic expenditure of singing in the species Teleogryllus commodus is approximately four times higher than that of resting [16]. Wing stroke rate is the main factor determining energetic costs of song [17], but only 0.05% of metabolic energy is converted into acoustic energy [16]. The motor activity underlying silent singing is thus likely to incur almost all the energetic expense of sonorous signalling, but without any sexually selected benefit, illustrating the indirect costs that can affect individuals during early stages of evolutionary trait loss. In T. oceanicus, secondary mutations that mitigate energetic costs by reducing long-term calling effort in flatwing males are theoretically possible, but would need to either co-segregate with flatwing-causing mutation(s), or be sufficiently beneficial to counterbalance selection against them when expressed in normal-wing males. Costs of silent singing might be particularly likely to impose selection for reduced calling effort in populations where flatwing males predominate.

Second, silent singing in flatwing T. oceanicus provides a counter-example to the frequent observation that behavioural resistance—adaptive behavioural change under stressful conditions—underlies rapid adaptation to ecological or environmental pressures [18] (e.g. escape behaviour in the lizard Anolis sagrei [19] and parasite tolerance in the frog Hyla femoralis [20]). In T. oceanicus, adaptation occurred through morphological, not behavioural, change. Although behaviour has been suggested to facilitate rapid evolution by enabling plasticity or relaxing selection [21], thereby accommodating indirect fitness costs of adaptive mutations, the persistence of silent singing in T. oceanicus highlights the need to also test behaviour's inhibitory effects on evolutionary adaptation.

Finally, remnants of lost sexual traits may represent a particularly evolvable substrate upon which selection can act. Patterned wing movements specific to long-range calling in T. oceanicus are not known to serve any other function, and it is unlikely that the air currents and substrate vibrations they create are detectable over longer distances given their rapid attenuation [22]. Flatwing males still attempt to produce courtship song, but this is also silent [23]. Over longer evolutionary timescales, co-option of vestigial signal components for different functions might represent a path of least resistance to the acquisition of evolutionary novelties. Recent evidence that vibration-duetting courtship behaviour of Lebinthine crickets may have arisen from a behaviour originally used for predator avoidance is consistent with this idea [5]. Future work testing whether vestigial trait components respond to different selective pressures following the loss of their original function could ultimately illuminate mechanisms by which evolutionary novelties arise.

Ethics

The species used in this study is not subject to ethical review, but we complied with ASAB's guidelines for the use of animals in research.

Data accessibility

Data are available in the Dryad Digital Repository (http://dx.doi.org/10.5061/dryad.7tv59) [24].

Authors' contributions

N.W.B. and B.H. conceived the study. W.T.S. collected and analysed the data. All authors contributed to experimental design, interpretation of results, and writing. All authors approve the final manuscript and agree to be held accountable for its content.

Competing interests

We declare we have no competing interests.

Funding

This research was supported by Natural Environment Research Council grants to N.W.B. (NE/L011255/1 and NE/I027800/1).

Acknowledgements We thank Sonia Pascoal for establishing cricket lines, Audrey Grant and Meghan McGunnigle for cricket husbandry, and Michael Ritchie for lending us an ultrasound microphone.

Footnotes

Electronic supplementary material is available online at https://dx.doi.org/10.6084/m9.figshare.c.3990303.